cellulose

0 downloads 0 Views 5MB Size Report
May 10, 2017 - polyurethane nanocomposites were used to prepare foamed ..... (A) DSC results for PU/CNWs; (B) Water absorption of PU/CNWs over a period ...
Carbohydrate Polymers 171 (2017) 281–291

Contents lists available at ScienceDirect

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

Research Paper

Preparation and evaluation of polyurethane/cellulose nanowhisker bimodal foam nanocomposites for osteogenic differentiation of hMSCs Ehsan Shahrousvand a , Mohsen Shahrousvand b , Marzieh Ghollasi c , Ehsan Seyedjafari d , Iman Sahebi Jouibari e , Amir babaei f , Ali Salimi a,∗ a

Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran Young Researchers and Elites Club, Science and Research Branch, Islamic Azad University, Tehran, Iran c Department of Cell and Molecular Biology, Faculty of Biological Science, Kharazmi University, Tehran, Iran d Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran e Department of Polymer Engineering & Color Technology, Amirkabir University of Technology, Tehran, Iran f Polymer Engineering Department, Faculty of Engineering, Golestan University, 4918888369, Gorgan, Iran b

a r t i c l e

i n f o

Article history: Received 12 December 2016 Received in revised form 24 April 2017 Accepted 7 May 2017 Available online 10 May 2017 Keywords: Polyurethane (PU) Cellulose nanowhisker (CNW) Human mesenchymal stem cell (hMSC) Osteogenic differentiation Bimodal foam scaffold

a b s t r a c t Biocompatible and biodegradable polyurethanes (PUs) based on polycaprolactone diol (PCL) were prepared and filled with cellulose nanowhiskers (CNWs) obtained from wastepaper. The incorporated polyurethane nanocomposites were used to prepare foamed scaffolds with bimodal cell sizes through solvent casting/particulate leaching method. Sodium chloride and sugar porogens were also prepared to fabricate the scaffolds. The mechanical and thermal properties of PU/CNW nanocomposites were investigated. Incorporation of different CNWs resulted in various structures with tunable mechanical properties and biodegradability. All bimodal foam nanocomposites were biodegradable and also noncytotoxic as revealed by MTT assay using SNL fibroblast cell line. PU/CNW foam scaffolds were used for osteogenic differentiation of human mesenchymal stem cells (hMSCs). Based on the results, such PU/CNW nanocomposites could support proliferation and osteogenic differentiation of hMSCs in threedimensional synthetic extracellular matrix (ECM). © 2017 Elsevier Ltd. All rights reserved.

1. Introduction As a new and promising interdisciplinary field, tissue engineering aims at regenerating injured tissues and organs via replacing them with engineered tissues using appropriate materials (Lai, Shalumon, Chen, & Chen, 2014; Lanza, Langer, & Vacanti, 2011). Artificial tissues are prepared to restore the normal functions of damaged tissues during the regeneration process and subsequently integrate with the host tissue. In this regard, treatment of bone defects using osteoconductive materials, osteogenic cells and osteoinductive molecules has been extensively studied (Cornejo et al., 2012; Ratanavaraporn, Kanokpanont, Tabata, & Damrongsakkul, 2009). Tissue engineering includes three fundamental components: cells, degradable scaffold and growth factors. In bone tissue engineering, scaffolds should facilitate cell adher-

∗ Corresponding author at: Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. P.O. Box 19945-546, Tehran, Iran. Tel.: +98 21 82482444; fax: +98 21 88053609. E-mail addresses: [email protected], [email protected] (A. Salimi). http://dx.doi.org/10.1016/j.carbpol.2017.05.027 0144-8617/© 2017 Elsevier Ltd. All rights reserved.

ence, proliferation and osteogenic differentiation and fully the formation of new bone tissues. Hence, a suitable scaffold should provide appropriate cell-material interactions and adequate pore size and porosity for the proper diffusion of nutrients into, and wastes out of such structure (Grenier, Sandig, & Mequanint, 2007; Shahrousvand, Sadeghi, & Salimi, 2016). An ideal scaffold should also provide the required mechanical support according to the desired tissue and resemble the extracellular matrix (ECM) to support the survival, proliferation, and differentiation of the seeded cells. Different materials such as metals, natural and synthetic polymers, minerals, etc. have been used to fabricate scaffolds (Hutmacher, 2000; Sabir, Xu, & Li, 2009; Stevens, Yang, Mohandas, Stucker, & Nguyen, 2008). Polymeric scaffolds, which can be easily designed, have been extensively used in bone tissue engineering (Dhandayuthapani, Yoshida, Maekawa, & Kumar, 2011; Sabir et al., 2009). Polyurethanes (PUs) with extensive diversity of structure and property, are among the most biocompatible materials and have been used for various biomedical applications due to their attractive physical and mechanical properties (Zdrahala & Zdrahala, 1999). PUs are a wide family of segmented block copolymers com-

282

E. Shahrousvand et al. / Carbohydrate Polymers 171 (2017) 281–291

posed of macrodiols as the soft segment (SS), diisocyanates and low molecular weight chain extender diols or diamines as the hard segment (HS) (Shahrousvand, Mir Mohamad Sadeghi, & Salimi, 2016). Biodegradable polyurethane scaffolds have been previously investigated for skin, cartilage, bone, nerve and cardiovascular tissue regeneration (Gorna & Gogolewski, 2006; Guan, Fujimoto, Sacks, & Wagner, 2005; Niu et al., 2014; Priya, Jungvid, & Kumar, 2008). An ideal polymeric scaffold should support cell attachment and ingrowth of new tissue, and biodegrade at a rate matching that of the formation of new tissue (Li et al., 2009). Various methods have been proposed to prepare porous scaffolds, including electrospinning, freeze drying, liquid–liquid and liquid-solid phase inversion, thermally induced phase separation, gas foaming and solvent casting/particulate leaching. However, each method is associated with its own advantages and drawbacks (Weigel, Schinkel, & Lendlein, 2006). Providing the required mechanical support by a porous scaffold which contains a large number of holes in its structure is a challenging task. To overcome this problem, the properties of polymeric scaffolds have been improved through dispersion of reinforcing materials into the polymer matrix with various concentrations (Ramakrishna, Mayer, Wintermantel, & Leong, 2001; Rezwan, Chen, Blaker, & Boccaccini, 2006). Cellulose nanowhiskers (CNWs) have been frequently applied as reinforcing materials in many scientific fields due to their unique and amazing chemical and physical properties (Eichhorn, 2011; Khalil, Bhat, & Yusra, 2012). CNWs extracted from lignocellulosic microfibrils exhibit higher elastic modulus (150 GPa) compared to S-glass (85 GPa) and aramid fibers (65 GPa) which have attracted great deal of attention for high performance applications (Saïd Azizi Samir, Alloin, Paillet, & Dufresne, 2004). Cellulose based nanobiocomposites as a promising class of engineered materials is derived from natural and synthetic polymers and organic/inorganic fillers at the nanoscale. Hence, nanobiocomposites can be used in tissue engineering and biomedical applications (Liu et al., 2016; Siqueira, Bras, & Dufresne, 2010). In this work, the solvent casting/particulate leaching method was used to prepare novel PU/CNW scaffolds based on cellulose nanocrystals extracted from wastepaper and polyurethane as the matrix. Different characterization methods were used to compare the effects of inclusion and dispersion of CNWs on the physic-chemical, mechanical, morphological and biocompatibility properties of the scaffolds. Finally, the osteogenic differentiation of hMSCs cultured on the prepared scaffolds was investigated. The PU/CNW bimodal foam nanocomposites can provide new insights into the bone tissue engineering.

2. Experimental 2.1. Materials According to our previous work, polycaprolactone diol (PCL) (Mn = 2000 Da, PDI = 1.04) was synthesized and dried in a threenecked flask under vacuum and magnetic stirring at 100 ◦ C for 120 min (Shahrousvand, Mir Mohamad Sadeghi, Salimi, & Nourany, 2016). Hexamethylene diisocyanate (HDI), 1, 4-Butanediol (BDO), N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO) were purchased from Merck (Germany) and used as received. Phosphate buffered saline (with an approximate pH of 7.3) was obtained from Gibco company. Fibroblast Cells (SNL76/7) and hMSCs were obtained from Stem Cell Technology Research Center (Iran) and cultured in a T-75 culture flask. (3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide) (MTT) was purchased from Sigma. Paper powders from Linter Pak Co (Iran) were used as microcrystalline cellulose. Phosphoric acid (85% H3 PO4 – ortho-Phosphoric acid) was purchased from Merck Mil-

lipore. Double-distilled deionized water was used at various stages of the work. 2.2. Preparation of cellulose nanowhiskers Cellulose nanocrystals were prepared from wastepaper. Cellulose microfibers (10 g – based on dry weight) were hydrolyzed in 190 ml of phosphoric acid solution (85 wt%) under strong agitation at 80 ◦ C for 30 min. Hydrolysis was terminated by adding 300 ml of cold water. The diluted suspension was centrifuged at 10,000 rpm for 5 min and the obtained precipitate was re-suspended in water with strong agitation, followed by centrifugation. This process was repeated until the pH of suspension reached 5. Thereafter, dialysis was carried out for 3 days until the pH became constant at 5.5–6. Subsequently, the suspension was dried at 40 ◦ C for 24 h and sonicated so as to disperse the nanofibers in DMF using an ultrasonic homogenizer at 19.5 kHz and 300 W output power (26 mm probe tip diameter, US-300T, Nissei, Japan) for 5 min to further enhance CNW extraction yield. 2.3. Synthesis of polyurethane PU was prepared using our previously reported method without using any catalysts (Shahrousvand, Mir Mohamad Sadeghi, & Salimi, 2016). Briefly, PCL was added to a glass vial reactor (50 ml) as the macro diol and dehydrated under reduced pressure at nearly 100 ◦ C for 2 h and then, the temperature was reduced to 80 ◦ C. PCL (12 g) reacted with HDI (3.02 g) at 80 ◦ C (the overall ratio of NCO to OH was kept at 3:1) using a magnetic heater-stirrer under partial vacuum without a reflux condenser. The reaction continued until NCO content reached the theoretical value which was determined via dibutyl amine titration according to ASTM D 2572. The time required for completion of the reaction between isocyanate groups and the two ends of macro diols was 3 h. Then BDO (1.08 g) was added to the synthesized pre-polymers until the reactor contents became viscose. Thereafter, reactor contents were drained into a Teflon petri dish and placed in an oven at 80 ◦ C for 24 h. 2.4. Preparation of interconnected bimodal foams PU/CNW To fabricate traditional salt-leached scaffolds, PU (2 g) with various contents of CNW filler (0%, 0.1%, 0.5% and 1% wt. related to polymer) and sugar (5% wt. related to polymer) was dissolved in 15 ml of DMF at 70 ◦ C. To prepare bimodal foam scaffolds with improved interconnectivity, To prepare bimodal foam scaffolds with improved interconnectivity, the PU/CNW/sugar dispersion was mixed with an equivalent amount of NaCl salt (with an average crystal size of 100–250 ␮m) with a weight ratio of 10:2. The mixed viscous suspension was transferred to a Teflon mold and placed in a laminar flow hood for at least 2 days for solvent evaporation. Thereafter, the salt and sugar were leached out through immersion in deionized water for 72 h, with replacing the water every 8 h. Finally, PU/CNW foams were immersed in PBS solution overnight before the subsequent cell seeding. 2.5. Characterization 2.5.1. Fourier-transformed infrared spectra analysis Fourier transform infrared spectroscopy (FTIR) was performed using an Equinox 55 FTIR spectrometer with 100 scans for a wave length range of 400–4000 cm−1 . 2.5.2. X-ray diffraction analysis The crystal structures of PU and PU/CNWs were determined via X-ray diffraction (XRD) analysis using a Rigaku RINT 2000 at 20 kV and 2 mA.

E. Shahrousvand et al. / Carbohydrate Polymers 171 (2017) 281–291

2.5.3. Mechanical analysis Tensile mechanical properties were examined by an Instron tensile testing apparatus (5566-Applied Science Co., Ithaca, NY). Tensile strength and elongation were measured at a crosshead speed of 10 mm/min. Young’s modulus of elasticity (using a linear region of 0–5% elongation), ultimate tensile strength and elongation at break were derived from the stress-strain curves. 2.5.4. Morphology observation and porosity measurement The morphologies of fabricated bimodal foam nanocomposites and their cell adherence were examined using a scanning electron microscope (SEM, KYKY-EM3200, Japan) after gold coating at an operating voltage of 26 KV. Cell morphology was photographed by a Leica Leitz optical microscope (OM) (Leica Inc., Foster City, CA) during the evaluation of cell viability on different days. The porosity of bimodal foams was measured using a specific gravity bottle based on Archimedes’ Principle (Yang, Shi et al., 2002). Briefly, scaffold porosity was determined as the following: Porosity (%) =

(W2 − W3 − Ws ) /e (W1 − W3 ) /e

(1)

where, W1 , W2 , W3 , Ws and ␳e denote the weight of specific gravity bottle filled with ethanol, the weight of specific gravity bottle containing ethanol and scaffold, the weight of specific gravity bottle from which the ethanol-saturated scaffold has taken out scaffold weight and ethanol density, respectively. 2.5.5. Water uptake of PU/CNWs The water sorption of samples was measured by a weighting method under equilibrium conditions at 25 ◦ C. Samples were placed as 20 mm × 30 mm thick strips in 30 ml of distilled water at 37 ◦ C. The water content (%) was calculated as the following: water uptake =

(Wt − W0 ) × 100 W0

(2)

where Wt and W0 are the maximum wet weight measured at time “t” and the dry weight of sample, respectively. 2.5.6. Biodegradation of PU/CNW bimodal foams The accelerated hydrolytic degradation tests were performed under basic conditions by placing the scaffolds into small containers with 15 ml of 1 mol/L NaOH and transferring them to an oven at 60 ◦ C for time periods of 2, 4, 10, 24 and 48 h, after which the scaffolds washed with distilled water and dried in a vacuum oven at 70 ◦ C for 24 h. The remaining mass was calculated according to Eq. (3). Remaining Mass (%) = 100 −



(Mi − Mf ) × 100 Mi

 (3)

where Mi and Mf denote the initial sample mass and the mass after degradation, respectively. The simulated body fluid (SBF) solution was prepared by dissolving NaCl (0.1368 mol), NaHCO3 KCl (0.003 mol), K2 HPO4 ·3H2 O (0.0013 mol), (0.0041 mol), MgCl2 ·6H2 O (0.0032 mol), CaCl2 (0.002 mol) and Na2 SO4 (0.0005 mol) (obtained from Merck) in 1 l of distilled water and buffering at a pH of 7.4 by adjusting HCl volume (Merck) at 37 ◦ C (Shahrousvand, Hoseinian et al., 2016). PU/CNW samples were weighed and placed in vials containing approximately 20 ml of the SBF solution and incubated at 37 ◦ C. Degradation was observed for 48 days. Samples were removed at different time intervals, washed with distilled water and dried in a vacuum oven at 70 ◦ C for 24 h. The remaining weight of the foams was evaluated based on mass loss according to Eq. (3).

283

2.5.7. Cell culture SNL76/7 cells and hMSCs were cultured in Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) penicillin G sodium (10 units/ml) and streptomycin sulfate (10 mg/ml), all purchased from Gibco BRL (NY, USA) in a humidified atmosphere of 5% CO2 at 37 ◦ C. The culture medium was replaced every 48 h until cells reached 80% confluency. Cells at the second passage were used for the study. 2.5.8. Osteogenic differentiation For osteogenic induction of hMSCs, they were incubated with osteogenic medium, containing DMEM supplemented with 10% FBS, 50 mg/ml ascorbic acid 2-phosphate (Sigma), 10 nM dexamethasone (Sigma) and 10 mM ␤-glycerophosphate (Sigma) at 37 ◦ C and 5% CO2 for two weeks. 2.5.9. Cell viability assays The proliferation of fibroblast cells on different scaffolds was evaluated via MTT assay (Shahrousvand, Mir Mohamad Sadeghi, & Salimi, 2016). Sterilized PU/CNW samples were placed in a 24-well culture plate, seeded with a cell density of 1 × 104 cells per well and incubated at 37 ◦ C and 5% CO2 . After 2, 4 and 6 days of cell culture, 50 ml of MTT solution (5 mg/ml) was added to each well. Then the plate was incubated at 37 ◦ C for 3 h. Thereafter, the supernatant was removed and a constant amount of an appropriate solvent was added. The optical density was read by a micro-plate reader (BioTek Instruments, USA) at a wavelength of range 570–630 nm. The same procedure was performed for cultured cells in tissue culture polystyrene (TCPS) as the control. Then the viability was calculated using the following equation: Cell Viability (%) =

OD × 100 OD0

(4)

where OD and OD0 represent the optical densities of the cell cultured nanoparticle suspensions and the blank control, respectively. Cell viability of PU/CNW samples was evaluated through Acridine Orange/ethidium bromide (AO/EtBr) staining. A fluorescent staining solution (1 ␮l) containing 100 ␮g/ml AO and 100 ␮g/ml EtBr was added to each well which was then rinsed with PBS and investigated by a fluorescent microscope (Leica Inc., Foster City, CA). 2.5.10. Alkaline phosphatase assay ALP activity as a marker of osteogenesis was measured during osteogenic differentiation of hMSCs. On days 7 and 14, the plates were washed three times with PBS. Total proteins of cells in TCPS and wells containing PU/CNWs were extracted using 200 ␮l of radio immune precipitation (RIPA) lysis buffer. The lysate was then centrifuged at 15,000g for 15 min at 4 ◦ C to sediment cell debris. The supernatant was collected and ALP activity was measured with an ALP assay kit (Parsazmun, Tehran, Iran) (Amiri, Ghollasi, Shahrousvand, Kamali, & Salimi, 2016). The enzymatic activity (IU/l) was normalized against the total protein (mg). Fluorescence intensity was determined at a 480 nm excitation and a 520 nm emission using a micro-plate reader (BioTek Instruments, USA). 2.5.11. Calcium content assay The amount of calcium minerals deposited by hMSCs under osteogenic induction on TCPS and wells containing PU/CNWs was measured using a calcium assay kit (Parsazmun, Tehran, Iran) (Amiri et al., 2016). Calcium extraction was performed through homogenization of well contents in 0.6 mol/l hydrochloric acid (Merck) followed by shaking for 4 h at 4 ◦ C. Optical density was measured at 570 nm after reagent addition to the calcium solutions.

284

E. Shahrousvand et al. / Carbohydrate Polymers 171 (2017) 281–291

Fig. 1. (A) The proposed process for CNW extraction from wastepaper; (B) The proposed mechanism of the bulk polymerization of PCL based polyurethanes.

Fig. 2. (A) SEM image of cellulose microfibers. As it is evident, the average fiber diameter was about 30 ␮m. (B) SEM image of CNWs extracted via phosphoric acid hydrolysis protocol. (C) The diameter distribution of CNWs measured using the Digimizer software through drawing three middle lines for each fiber. (D) and (E) SEM images of porous PU/CNW-1 in two magnifications.

Calcium content was obtained from a standard concentration curve of serial dilutions of calcium versus the corresponding OD.

2.5.12. Alizarin red staining and mineralization To determine calcium deposition, alizarin red staining was performed on the 14th day to evaluate the mineralized matrix. The

E. Shahrousvand et al. / Carbohydrate Polymers 171 (2017) 281–291

285

Fig. 3. (A) FTIR spectra of the prepared PU/CNWs; (B) XRD patterns of PU/CNW nanocomposites; (C) Typical stress–strain curves of PU/CNWs and (D) Young’s Modulus and tensile strength of PU/CNWs.

medium was removed and cells were washed three times with PBS and fixed in 4% paraformaldehyde for 10 min at 4 ◦ C. These cells were then washed with PBS twice. The fixed samples were stained with 1% Alizarin red at pH 7.2 (Sigma) at room temperature. After 5–10 min, the cells were washed three times with PBS and examined by light microscopy. For von Kossa evaluation of cell mineralization, after 14 days of incubation with osteogenic medium, cells were fixed with 10% formalin and washed twice with double distilled water. The cells were stained with 5% silver nitrate solution and washed twice with double distilled water. They were then fixed with 5% sodium thiosulfate and wells were washed 3–4 times with double distilled water. The stained scaffolds were transferred to a fresh culture dish for subsequent imaging using light microscopy. 2.5.13. Statistical analysis Analysis of variance (one–way ANOVA – Post hoc analysis – the least significant difference test) was used to evaluate the significance of difference between the incubation days for cell proliferation, alkaline phosphatase activity and calcium content. Statistical significance level was set to 0.05 (*). All experiments were conducted at least 3 times and data were expressed as mean ± standard deviation (SD). 3. Result and discussion 3.1. Preparation of polyurethane nanocomposite bimodal foams Fig. 1A indicates a simplified process flow diagram related to the preparation of CNWs from the microcrystalline cellulose (MCC) fibers. Wastepaper fibers were mechanically shredded. After soaking MCC fibers in distilled water, MCC pulp was squeezed

to facilitate mixing with phosphoric acid. MCC fibers consisted of amorphous and crystalline regions, in which the amorphous regions were associated with lower densities compared to the crystalline regions. Thus, when cellulose fibers were hydrolyzed due to acid treatment, the amorphous regions broke up, releasing individual crystallites (Engström, Ek, & Henriksson, 2006). To separate nanoparticles from each other, cellulose nanocrystals were spread in DMF and sonicated so as to break aggregates. It should be noted that excessive sonication breaks the cellulose nanocrystals, leading to a decreased crystallinity of CNWs. However, it results in smaller particles and increases their surfaces. As shown in Fig. 1B, polyurethane was synthesized using a two-step bulk reaction through melting the pre-polymer method, utilizing 1, 4-BDO as the chain extender, HDI as the HS and PCL as the SS without any catalyst (Liu, Jiang, Shi, & Zhang, 2012). The HS wt.% in the polyurethane is related to the molar number and the molar masses were calculated as the following: %HS =

nMHDI + mMBDO nMHDI + kMPCL + mMBDO

(5)

where M indicates the average molecular weight of reagents (g/mol) and n, m and k denote the mole number of reagents. The HS content calculated for PU was about 25.5 wt.%. Fig. 2B illustrates SEM images of the CNWs extracted from wastepaper microcrystals (Fig. 2A) before sample preparation. As can be seen, the average particle size was about 60–90 nm (Fig. 2C). Moreover, Fig. 2D, E depict the SEM images of the bimodal foams prepared from PU/CNW-1 through solvent casting/particle leaching. Larger and small holes are related to salt crystals and sugar, respectively. Since sugar and polyurethane were both dissolved in DMF; small holes were uniformly distributed throughout the scaffold, improving the interconnection of scaffold pores. Highly

286

E. Shahrousvand et al. / Carbohydrate Polymers 171 (2017) 281–291

Fig. 4. (A) DSC results for PU/CNWs; (B) Water absorption of PU/CNWs over a period of time. Remaining mass of samples: (C) After degradation in 1 mol l−1 NaOH solution at 60 ◦ C for different periods of time and (D) after incubation in SBF solution at 37 ◦ C.

interconnected channels can facilitate cellular infiltration and nutrient delivery. The pore size and porosity of the prepared scaffolds ranged from ∼20 to ∼150 ␮m and 82%, respectively. The size of small pores created by sugar was in the range of about 500 nm to 3 ␮m. 3.2. Physico-chemical characterization of PU/CNW bimodal foams FTIR peaks (Fig. 3A) showed same chemical structure of each PU/CNW sample. These spectra revealed the presence of characteristic polyurethane bands, N H stretching vibration at 3330–3450 cm−1 , asymmetric and symmetric CH2 stretching at 2934 cm−1 and 2850 cm−1 , respectively, while other modes of CH2 vibrations were manifested by the bands at 1463, 1419, 1396, 1365 and 1295 cm−1 . Characteristic urethane bands were in the range 3330–3450 cm−1 . Characteristic bands for the soft segments could be observed at 2934 cm−1 , 2850 cm−1 and in the range of 1449–1463 cm−1 . The absorbance at 1164 cm−1 and 1096 cm−1 was attributed to ester bonds, and the C O C stretching vibration band appeared at 1039–1044 cm−1 . The free (non-hydrogen bonded) N H stretching band appeared as a weak shoulder absorbing at ∼3340 cm−1 . Based on FTIR spectra, the 1760–1600 cm−1 region was assigned to the urethane and PCL ester C O group. The carbonyl bands at 1720–1721 cm−1 and 1732 cm−1 could be assigned to free urethane and ester, respectively. To study the crystalline structure of PU/CNWs, XRD analysis was performed as shown in Fig. 3B. The 2␪ angle at 22.9 is corresponding to cellulose (Zhou et al., 2009) and cellulose peaks

cannot be clearly observed in PU/CNWs with low CNW contents (Park, Oh, & Kim, 2013). In polyurethanes, microphase separation or microphase mixing of the SS and HS depends on their composition and structural order. Therefore for all samples, the two peaks in the XRD data indicated a typical pattern for a low crystalline polyurethane (2␪ angle of 19 and 23), suggesting that these PU/CNWs have semi-crystalline structures as a result of microphase mixing. The peak intensity indicates the crystallinity level of sample and hence, the crystallinity of PU/CNWs decreased as the CNW content increased, implying that PU crystallization had been inhibited by CNW. However, the presence of cellulose nanocrystals increased the peak intensity at a 2␪ angle of 23 it as for example evident for PU/CNW-1 (Park et al., 2013; Pei, Malho, Ruokolainen, Zhou, & Berglund, 2011). 3.3. Mechanical properties of PU/CNW bimodal foams The effect of CNW content (wt.%) on the mechanical properties of PU/CNW foams has been demonstrated in Fig. 3C and D. Tensile strength was considerably increased from 79 to 112 kPa by adding only 0.1 wt.% CNW which could be due to the effective reinforcement and suitable dispersion of nanoparticles in the polymer matrix (Gao et al., 2012; Pei et al., 2011). Tensile strength increased with increasing nanoparticle content that could be due to the presence of a large number of active hydroxyl groups on the surface of CNWs which can take part in the hydrogen bonding with urethane groups of PUs, resulting in strong interfacial adhesion between the matrix and the filler (Park et al., 2013; Pei et al., 2011; Rueda et al.,

E. Shahrousvand et al. / Carbohydrate Polymers 171 (2017) 281–291

287

Fig. 5. Acridine orange staining as a cell viability analysis after in vitro culture for 2 days, which red cells indicates they are undergoing apoptosis: (A) porous PU/CNW scaffold before cell seeding. SNLs cultured on (B) blank well as TCPS; (C) PU-CNW-0; (D) PU-CNW-0.1; (E) PU-CNW-0.5; (F) PU-CNW-1. (scale bar: 100 ␮m).

2013; Saralegi, Gonzalez, Valea, Eceiza, & Corcuera, 2014). Furthermore, increasing the percentage of CNW in polyurethane matrix resulted in a decrease in elongation at break and an increase in the Young’s modulus. The moduli of various body tissues are different together, such as the brain, muscle, and bone modulus being 1, 8–17, and more than 100 kPa, respectively (Choi, Kuhn, Ciarelli, & Goldstein, 1990; Currey, 2004, 2014; Reilly, Burstein, & Frankel, 1974). Cells sense matrix stiffness by a mechano-transducer and prepare their morphology and specific lineage by transducing this information to a nuclear cell. Cell shapes in different stiffness surfaces (brain, muscle, and bonelike) are branched shape, spindle shape, and the polygonal shape, respectively (Saha et al., 2008). With this backdrop, mechanical properties of scaffolds can affect cell fates.

3.4. Thermal study of PU/CNW bimodal foams Thermal properties of PU/CNWs were studied to investigate the effects of CNWs on the thermal behavior of PU. The existing incompatibility between the two segments leads to their separation into microphases. As presented in Fig. 4A, DSC thermographs showed that each sample had one glass transition temperature (Tg ) and two melting temperatures. So, thermal transitions revealed the microphase separation of the SS from the HS. All PU/CNWs showed similar temperature transitions and endotherms. In addition, the observed increasing trend of Tg of PU/CNWs with increasing CNW content could be attributed to the amorphous HS interactions as well as the hydrogen bonding of amorphous HS chains with nanoparticles. The heat of fusion decreased as the CNW content increased, suggesting the inhibition of PU crystallization by CNW. An increase in the CNWs loading led to shallower endothermic curves, indicating that the dispersed nanoparticles not only acted as seeds and speeded up the nucleation, but also played part as barriers to form large crystallites. In consistence with the presented

results, an increase in the CNW loading led to a decrease in chain mobility and subsequent enhancement in the Tg levels of PU/CNWs. 3.5. Water sorption and hydrophilicity of PU/CNW bimodal foams Wettability plays a critical role in the development of materials for application in the tissue engineering. The results of swelling in water have been shown in Fig. 4B. The amount of adsorbed water increased with increasing nanoparticle content in the nanocomposite membranes. This could be explained by the enhanced hydrophilicity of cellulose nanoparticles incorporated into PU scaffolds. Water penetration increased by increasing the CNW ratio in polyurethane structures. The hydrolytic degradation of ester-based polyurethanes involves three stages. The first is the incubation stage, in which the absorption of water occurs. The second is the induction stage, during which the polymer chain is broken via ester bonds. Finally, there is the erosion stage (Barrioni, de Carvalho, Oréfice, de Oliveira, & de Magalhães Pereira, 2015). Therefore, it is reasonable that polymer degradation is greater with increasing amounts of CNW concentration in the polyurethane structure. Water sorption is a key requirement of the scaffolds and improves biocompatibility and cell behavior (Yang, Bei, & Wang, 2002). 3.6. Biodegradability of PU/CNW bimodal foams Hydrolytic degradation is an importance issue in the design and procurement of materials for biomedical applications. Since in vivo degradation is a time consuming and laborious process, some techniques are applied to speed up the process (Barrioni et al., 2015). The degradation process can be accelerated in two ways: increasing the temperature of the degradation medium, and the addition of hydroxyl ions. Fig. 4C shows the accelerated degradation of PU/CNWs after different periods of time in 1 mol l−1 NaOH at 60 ◦ C. To verify and compare the results of high-speed degradation,

288

E. Shahrousvand et al. / Carbohydrate Polymers 171 (2017) 281–291

Fig. 7. SEM micrograph of attached and differentiated hMSCs on PU/CNW bimodal foams.

were green under a fluorescent light and there is no red cells in any of the images (Shahrousvand, Hoseinian et al., 2016). Results have been shown in Fig. 5A–F in which the seeded cells and their spreading into the prepared bimodal foam scaffolds can be observed. Fig. 6A shows the results of MTT assay for PU/CNWs after 2, 4 and 6 days of incubating SNLs cultures with the samples. All PU/CNW samples were associated with cell viabilities above 90%, being indicative of the non-cytotoxicity of all samples, although lower contents of CNW in nanocomposites resulted in better non-toxicity properties. Cytotoxicity can be classified based on cell viability relative to controls, as the activity relative to controls being less than 30% (severe cytotoxicity), between 30 and 60% (moderate cytotoxicity), and between 60% and 90% (slight cytotoxicity) or greater than 90% (not cytotoxic) (Barrioni et al., 2015). Therefore, the obtained results were indicative of the excellent biocompatibility of the reinforced polyester based polyurethane foams. 3.8. Alkaline phosphatase activity measurement

Fig. 6. (A) MTT assay using fibroblast cells (SNL76/7) cultured on PU/CNWs for 2, 4 and 6 days, in which a culture without any membrane was used as the blank control group; (B) ALP expression of hMSCs indicating the osteogenic differentiation after 7 and 14 days of culture (p < 0.05); (C) The measured optical density of calcium minerals deposited on TCPS and PU/CNWs by hMSCs under osteogenic induction (at 570 nm).

hydrolytic degradation in SBF has been shown in Fig. 4D. The highest degradation level was observed for PU-CNW-1 after 48 days as a result of the high CNW concentration in the polyurethane structure. The remaining mass after 48 h of degradation in NaOH solution was 72.6%, 70.1%, 68.3% and 64.2% for the PU-CNW-0, PU-CNW-0.1, PU-CNW-0.5 and PU-CNW-1 samples, respectively. 3.7. Cell viability and cytotoxicity of PU/CNW bimodal foams To determine cell viability on the nanocomposites, cells were cultured onto the samples for 2 days, washed with PBS and stained with 0.01% (w/v) acridine orange for 5 min. The nuclei of living cells

7 and 14 days after seeding hMSCs into PU/CNWs and incubating them with osteogenic medium, alkaline phosphatase (ALP) as a biochemical marker for osteoblast phenotype was stained according to a standard procedure suggested by the manufacturer instructions (Fig. 6B). Osteoblasts secrete alkaline phosphatase, which degrades inorganic pyrophosphates and causes an increase in phosphate levels thereby activating the mineralization process (Hosseinkhani, Hosseinkhani, Tian, Kobayashi, & Tabata, 2006). Therefore, ALP activity was considered as a direct measure of the functional activity of osteoblasts. ALP activity levels in the presence of PU/CNWs after 14 days were not significantly different compared to other samples. The results of ALP activity indicated that the reinforced polyurethane bimodal foams supported osteoblastic differentiation of hMSCs. 3.9. Calcium content assay In addition to ALP activity, mineralized cells were clearly identified by calcium deposition measured in differentiated hMSCs on days 7 and 14 of osteogenic induction (Fig. 6C). Compared to other groups, the highest mineralization was detected in wells containing PU/CNWs. Fig. 7 shows SEM micrographs of induced hMSCs after 14 days of incubation with in differentiation medium. As can be

E. Shahrousvand et al. / Carbohydrate Polymers 171 (2017) 281–291

289

Fig. 8. Von Kossa staining confirmed the osteogenic differentiation of hMSCs. Cells cultured on (A) TCPS; (B) PU-CNW-0; (C) PU-CNW-0.1; (D) PU-CNW-0.5 and (E) PU-CNW-1 after 14 days. Alizarin red staining was used to confirm the osteogenic differentiation of hMSCs. Cells cultured on (F) TCPS; (G) PU-CNW-0; (H) PU-CNW-0.1; (I) PU-CNW-0.5 and (J) PU-CNW-1 after 14 days (scale bar: 200 ␮m).

290

E. Shahrousvand et al. / Carbohydrate Polymers 171 (2017) 281–291

seen, hMSCs appropriately proliferated, well spread and attached to the bimodal foam PU/CNWs. 3.10. Mineralization and alizarin red staining The results of von Kossa staining which was used to detect the mineralization in PU/CNW scaffolds was more intense in PU/CNW scaffolds compared to control wells. After 14 days, deep blackish green von Kossa stains were observed in PU/CNW scaffolds and less intense stains were observed in TCPS (Fig. 8A–E). After 14 days of culture, the alizarin red staining was deeper in PU/CNWs when compared to TCPS (Fig. 8F–J). Alizarin red and von Kossa staining confirmed the osteogenic differentiation of hMSCs on PU/CNW bimodal foam scaffolds. The presence of phosphate structures on CNWs can affect osteogenic differentiation of stem cells (de Jonge et al., 2010). 4. Conclusion The aim of this study was to fabricate bimodal nanocomposite foams based on PU reinforced by cellulose nanocrystals and to investigate their potential to support the adhesion, proliferation and osteogenic differentiation of hMSCs. Tensile strength and elastic modulus of highly porous PU/CNW nanocomposites were effectively increased with CNW addition, because of the reinforcing effect of stiff nanowhiskers in the rubbery matrix in association with the strong interfacial adhesion between PU and CNW surfaces. Various assessments such as ALP activity, calcium content, alizarin red, mineralization staining and SEM micrographs demonstrated that these interconnected porous nanocomposites are appropriate for hMSCs osteogenesis. Financial & competing interests disclosure This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgments We acknowledge Ali Karbalaeimahdi and Maryam Jafari for their helpful comments. The spiritual results of this work are dedicated to bone disease-patients. References Amiri, B., Ghollasi, M., Shahrousvand, M., Kamali, M., & Salimi, A. (2016). Osteoblast differentiation of mesenchymal stem cells on modified PES-PEG electrospun fibrous composites loaded with Zn 2 SiO 4 bioceramic nanoparticles. Differentiation, 92(4), 148–158. Barrioni, B. R., de Carvalho, S. M., Oréfice, R. L., de Oliveira, A. A. R., & de Magalhães Pereira, M. (2015). Synthesis and characterization of biodegradable polyurethane films based on HDI with hydrolyzable crosslinked bonds and a homogeneous structure for biomedical applications. Materials Science and Engineering: C, 52, 22–30. Choi, K., Kuhn, J. L., Ciarelli, M. J., & Goldstein, S. A. (1990). The elastic moduli of human subchondral, trabecular, and cortical bone tissue and the size-dependency of cortical bone modulus. Journal of Biomechanics, 23(11), 1103–1113. Cornejo, A., Sahar, D. E., Stephenson, S. M., Chang, S., Nguyen, S., Guda, T., . . . & Sharma, R. (2012). Effect of adipose tissue-derived osteogenic and endothelial cells on bone allograft osteogenesis and vascularization in critical-sized calvarial defects. Tissue Engineering Part A, 18(15–16), 1552–1561. Currey, J. D. (2004). Tensile yield in compact bone is determined by strain, post-yield behaviour by mineral content. Journal of Biomechanics, 37(4), 549–556. Currey, J. D. (2014). The mechanical adaptations of bones. Princeton University Press. de Jonge, L. T., Leeuwenburgh, S. C., van den Beucken, J. J., te Riet, J., Daamen, W. F., Wolke, J. G., . . . & Jansen, J. A. (2010). The osteogenic effect of electrosprayed nanoscale collagen/calcium phosphate coatings on titanium. Biomaterials, 31(9), 2461–2469.

Dhandayuthapani, B., Yoshida, Y., Maekawa, T., & Kumar, D. S. (2011). Polymeric scaffolds in tissue engineering application: A review. International Journal of Polymer Science, 2011. Eichhorn, S. J. (2011). Cellulose nanowhiskers: Promising materials for advanced applications. Soft Matter, 7(2), 303–315. Engström, A.-C., Ek, M., & Henriksson, G. (2006). Improved accessibility and reactivity of dissolving pulp for the viscose process: Pretreatment with monocomponent endoglucanase. Biomacromolecules, 7(6), 2027–2031. Gao, Z., Peng, J., Zhong, T., Sun, J., Wang, X., & Yue, C. (2012). Biocompatible elastomer of waterborne polyurethane based on castor oil and polyethylene glycol with cellulose nanocrystals. Carbohydrate Polymers, 87(3), 2068–2075. Gorna, K., & Gogolewski, S. (2006). Biodegradable porous polyurethane scaffolds for tissue repair and regeneration. Journal of Biomedical Materials Research Part A, 79(1), 128–138. Grenier, S., Sandig, M., & Mequanint, K. (2007). Polyurethane biomaterials for fabricating 3D porous scaffolds and supporting vascular cells. Journal of Biomedical Materials Research Part A, 82(4), 802–809. Guan, J., Fujimoto, K. L., Sacks, M. S., & Wagner, W. R. (2005). Preparation and characterization of highly porous, biodegradable polyurethane scaffolds for soft tissue applications. Biomaterials, 26(18), 3961–3971. Hosseinkhani, H., Hosseinkhani, M., Tian, F., Kobayashi, H., & Tabata, Y. (2006). Osteogenic differentiation of mesenchymal stem cells in self-assembled peptide-amphiphile nanofibers. Biomaterials, 27(22), 4079–4086. Hutmacher, D. W. (2000). Scaffolds in tissue engineering bone and cartilage. Biomaterials, 21(24), 2529–2543. Khalil, H. A., Bhat, A., & Yusra, A. I. (2012). Green composites from sustainable cellulose nanofibrils: A review. Carbohydrate Polymers, 87(2), 963–979. Lai, G.-J., Shalumon, K., Chen, S.-H., & Chen, J.-P. (2014). Composite chitosan/silk fibroin nanofibers for modulation of osteogenic differentiation and proliferation of human mesenchymal stem cells. Carbohydrate Polymers, 111, 288–297. Lanza, R., Langer, R., & Vacanti, J. P. (2011). Principles of tissue engineering. Academic press. Li, B., Yoshii, T., Hafeman, A. E., Nyman, J. S., Wenke, J. C., & Guelcher, S. A. (2009). The effects of rhBMP-2 released from biodegradable polyurethane/microsphere composite scaffolds on new bone formation in rat femora. Biomaterials, 30(35), 6768–6779. Liu, Q., Jiang, L., Shi, R., & Zhang, L. (2012). Synthesis, preparation, in vitro degradation, and application of novel degradable bioelastomers—A review. Progress in Polymer Science, 37(5), 715–765. Liu, M., Zheng, H., Chen, J., Li, S., Huang, J., & Zhou, C. (2016). Chitosan-chitin nanocrystal composite scaffolds for tissue engineering. Carbohydrate Polymers, 152, 832–840. Niu, Y., Chen, K. C., He, T., Yu, W., Huang, S., & Xu, K. (2014). Scaffolds from block polyurethanes based on poly (␧-caprolactone)(PCL) and poly (ethylene glycol)(PEG) for peripheral nerve regeneration. Biomaterials, 35(14), 4266–4277. Park, S. H., Oh, K. W., & Kim, S. H. (2013). Reinforcement effect of cellulose nanowhisker on bio-based polyurethane. Composites Science and Technology, 86, 82–88. Pei, A., Malho, J.-M., Ruokolainen, J., Zhou, Q., & Berglund, L. A. (2011). Strong nanocomposite reinforcement effects in polyurethane elastomer with low volume fraction of cellulose nanocrystals. Macromolecules, 44(11), 4422–4427. Priya, S. G., Jungvid, H., & Kumar, A. (2008). Skin tissue engineering for tissue repair and regeneration. Tissue Engineering Part B: Reviews, 14(1), 105–118. Ramakrishna, S., Mayer, J., Wintermantel, E., & Leong, K. W. (2001). Biomedical applications of polymer-composite materials: A review. Composites Science and Technology, 61(9), 1189–1224. Ratanavaraporn, J., Kanokpanont, S., Tabata, Y., & Damrongsakkul, S. (2009). Growth and osteogenic differentiation of adipose-derived and bone marrow-derived stem cells on chitosan and chitooligosaccharide films. Carbohydrate Polymers, 78(4), 873–878. Reilly, D. T., Burstein, A. H., & Frankel, V. H. (1974). The elastic modulus for bone. Journal of Biomechanics, 7(3), 271IN9273–9272IN12275. Rezwan, K., Chen, Q., Blaker, J., & Boccaccini, A. R. (2006). Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials, 27(18), 3413–3431. Rueda, L., Saralegui, A., d’Arlas, B. F., Zhou, Q., Berglund, L. A., Corcuera, M., . . . & Eceiza, A. (2013). Cellulose nanocrystals/polyurethane nanocomposites. Study from the viewpoint of microphase separated structure. Carbohydrate Polymers, 92(1), 751–757. Saïd Azizi Samir, M. A., Alloin, F., Paillet, M., & Dufresne, A. (2004). Tangling effect in fibrillated cellulose reinforced nanocomposites. Macromolecules, 37(11), 4313–4316. Sabir, M. I., Xu, X., & Li, L. (2009). A review on biodegradable polymeric materials for bone tissue engineering applications. Journal of Materials Science, 44(21), 5713–5724. Saha, K., Keung, A. J., Irwin, E. F., Li, Y., Little, L., Schaffer, D. V., & Healy, K. E. (2008). Substrate modulus directs neural stem cell behavior. Biophysical Journal, 95(9), 4426–4438. Saralegi, A., Gonzalez, M. L., Valea, A., Eceiza, A., & Corcuera, M. A. (2014). The role of cellulose nanocrystals in the improvement of the shape-memory properties of castor oil-based segmented thermoplastic polyurethanes. Composites Science and Technology, 92, 27–33. Shahrousvand, M., Hoseinian, M. S., Ghollasi, M., Karbalaeimahdi, A., Salimi, A., & Tabar, F. A. (2016). Flexible magnetic polyurethane/Fe 2 O 3 nanoparticles as

E. Shahrousvand et al. / Carbohydrate Polymers 171 (2017) 281–291 organic-inorganic nanocomposites for biomedical applications: Properties and cell behavior. Materials Science and Engineering: C, http://dx.doi.org/10.1016/j. msec.2016.12.117 Shahrousvand, M., Mir Mohamad Sadeghi, G., & Salimi, A. (2016). Artificial extracellular matrix for biomedical applications: Biocompatible and biodegradable poly (tetramethylene ether) glycol/poly (␧-caprolactone diol)-based polyurethanes. Journal of Biomaterials Science, Polymer Edition, 27(17), 1712–1728. http://dx.doi.org/10.1016/j.msec.2016.12.117 Shahrousvand, M., Mir Mohamad Sadeghi, G., Salimi, A., & Nourany, M. (2016). Bulk synthesis of monodisperse and highly biocompatible poly (␧-caprolactone)-diol by transesterification side-reactions. Polymer-Plastics Technology and Engineering [just-accepted]. Shahrousvand, M., Sadeghi, G. M. M., & Salimi, A. (2016). The superficial mechanical and physical properties of matrix microenvironment as stem cell fate regulator. Advanced Surfaces for Stem Cell Research, 23. Siqueira, G., Bras, J., & Dufresne, A. (2010). Cellulosic bionanocomposites: A review of preparation, properties and applications. Polymers, 2(4), 728–765. Stevens, B., Yang, Y., Mohandas, A., Stucker, B., & Nguyen, K. T. (2008). A review of materials, fabrication methods, and strategies used to enhance bone regeneration in engineered bone tissues. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 85(2), 573–582.

291

Weigel, T., Schinkel, G., & Lendlein, A. (2006). Design and preparation of polymeric scaffolds for tissue engineering. Expert Review of Medical Devices, 3(6), 835–851. Yang, J., Bei, J., & Wang, S. (2002). Improving cell affinity of poly (d, l-lactide) film modified by anhydrous ammonia plasma treatment. Polymers for Advanced Technologies, 13(3–4), 220–226. Yang, J., Shi, G., Bei, J., Wang, S., Cao, Y., Shang, Q., . . . & Wang, W. (2002). Fabrication and surface modification of macroporous poly (l-lactic acid) and poly (l-lactic-co-glycolic acid)(70/30) cell scaffolds for human skin fibroblast cell culture. Journal of Biomedical Materials Research, 62(3), 438–446. Zdrahala, R. J., & Zdrahala, I. J. (1999). Biomedical applications of polyurethanes: A review of past promises, present realities, and a vibrant future. Journal of Biomaterials Applications, 14(1), 67–90. Zhou, Q., Malm, E., Nilsson, H., Larsson, P. T., Iversen, T., Berglund, L. A., & Bulone, V. (2009). Nanostructured biocomposites based on bacterial cellulosic nanofibers compartmentalized by a soft hydroxyethylcellulose matrix coating. Soft Matter, 5(21), 4124–4130.