polyvinyl formal composite with a double crosslinking interpenetrating network for multifunctional biomedical application

Composites Part B 121 (2017) 9e22

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Reprint of: Preparation of a novel sodium alginate/polyvinyl formal composite with a double crosslinking interpenetrating network for multifunctional biomedical application Yansen Wang, Yudong Zheng*, Wei He, Cai Wang, Yi Sun, Kun Qiao, Xinyi Wang, Lin Gao School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, People's Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 September 2016 Received in revised form 1 December 2016 Accepted 28 January 2017

In recent years, porous materials with good absorbent capacity, mechanical properties and biocompatibility, has gradually become a research focus in the biomedical fields. In this study, a novel double crosslinking sodium alginate (SA)/polyvinyl formal (PVF) composite with interpenetrating polymer network (IPN) structure was developed. The composite was prepared through the blending of a PVA gel with SA, and then the obtained mixture was crosslinked by formaldehyde (HCHO) and calcium ion (Ca2þ). The foamed composite formed by this process is hydrophilic and has a continuous porous structure. The chemical structure, micromorphology, mechanical and thermal properties, water absorbing capability and hydroexpansivity of the SA/PVF composites changed with the different proportion and distribution of SA. The formation of this special structure dramatically improved the thermal and mechanical properties as well as hydroexpansivity of the composites. Besides, SA/PVF composites were good for cell attachment and normal proliferation, which was revealed by CCK-8 assays and fluorescence microscopy on rat bone marrow derived mesenchymal stem cells (MSCs). The composite has broad application prospects and will be a promising candidate for biomedical applications, such as medical sponges, scaffolds for tissue engineering, surgical filling sponges, wound dressings and so on. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Polyvinyl formal Sodium alginate Double crosslinking Foamed composites

1. Introduction Porous materials have good absorbent properties, swelling capacity and 3D porous structure. In recent years, a large number of researchers have devoted themselves to producing new and well performing wound dressings or tissue engineering scaffolds through the synthesis and modification of porous materials [1e6]. However, the structure and properties of most porous materials exert obvious disadvantages in the practical application. For instance, the porosity of porous material is hard to control and the pore size is also difficult to regulate and control. Also, most porous materials have poor mechanical strength and biocompatibility [7], DOI of original article: http://dx.doi.org/10.1016/j.compositesb.2017.01.045. A publisher's error resulted in this article appearing in the wrong issue. The article is reprinted here for the reader's convenience and for the continuity of the special issue. For citation purposes, please use the original publication details; JCOMB, 114 (2017) 149-162. * Corresponding author. E-mail addresses: [email protected] (Y. Zheng), hewei881130@ 126.com (W. He), [email protected] (C. Wang), [email protected] (Y. Sun), [email protected] (K. Qiao), [email protected] (X. Wang), maria_213@163. com (L. Gao). http://dx.doi.org/10.1016/j.compositesb.2017.06.023 1359-8368/© 2017 Elsevier Ltd. All rights reserved.

which severely impacts the future industry application as well as medical application. Besides, most currently used porous materials have bad forming nature and they are difficult to process and use [8,9]. Ideal porous materials must possess certain mechanical strength, liquid-absorption ability, three-dimensional networks and good forming nature. Pore size should also be regulated and controlled in a wide range to adapt to different application environments. Through modification, the pore size of the porous materials can be made controllable, the mechanical properties and biocompatibility can be significantly improved as well, which will extend their application range. The modified porous materials are now in highly demand in the biomedical fields. As a traditional hydrophilic porous material, PVF foam exhibits good properties, for instance, open-cell structure, high water absorbing rate, and super softness at wet state [10]. It can be designed to achieve an applicable porosity for cellular infiltration, and suitable compliance for handling so as to be conformable with varying tissue and organ shapes [11,12]. Recent research has shown that, PVF composites have been used in polymer electrolytes [13,14], wastewater treatment [15], sound absorption [16] and some other industrial production areas. However, few studies have

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reported the biomedical use of modified PVF. The reason for the low usage rate as biomaterials is mainly due to that the modified PVF composite doesn't have adequate mechanical properties and its biocompatibility is quite insufficient. For instance, most modified PVF materials doesn't have adequate mechanical properties, the tensile strength only reaches 100e500 KPa and the abrasion resistance and compression strength cannot meet the requirements [17,18]. Common modified PVF materials are always made into resins, membranes, glues and slices [13,19], yet they don't have expansion capabilities. Hou et al. [7] have tested the cytocompatibility of modified PVF, the experimental results suggested that PVF materials had a bad biocompatibility and certain cytotoxicity. Besides, the pore size of the PVF porous composites is difficult to regulate and control. Togami et al. [19]prepared dextran-coated PVF with better biomechanical strength and osteogenic response. The obtained composites showed a good prospect as a fill material for bone defects. Miyoshi et al. [20] utilized modified porous polyvinyl formal to acquire biological responses and long-term stable structure in hepatocyte culture. However, in general, the performance of modified PVF has not been well improved as expected, which would influence its practicability and application range in the biomedical fields. There are many ways to improve the mechanical properties and biocompatibility of synthetic polymers, such as surface modification, physical blending and chemical grafting etc. Among these methods, IPN technology is an effective method for stable integration of two polymer networks with different properties or functions [21,22]. IPN exhibits good mechanical properties. Due to its large equilibrium adsorption capacity, high adsorption selectivity, diverse chemical structure and easy regeneration property, IPN is one of the most efficient adsorbents for fast absorbing exudate [23e26]. Utilizing porous polymer and polysaccharide to form an IPN composites may achieve improved mechanical properties and biocompatibility because of their highly entangled networks [27e29]. Sodium alginate is a polysaccharide obtained from brown seaweeds [30]. It has been extensively studied for a wide variety of biological applications due to its low cost, biocompatibility, biodegradability, ease of chemical derivatization and high rates of degradation [31]. Calcium ions have been used as a crosslinking agent to form alginate hydrogel in several studies [32]. As alginate hydrogels retain a structural similarity to the extracellular matrices in tissues, they have been particularly attractive in wound healing [33e35] and tissue engineering applications [36,37]. In this study, SA was blended with PVA and then crosslinked by HCHO and Ca2þ respectively. PVA could form a permanent network after crosslinked by HCHO while SA could form an “egg box” shaped network in the presence of Ca2þ, and so the obtained composites can be considered as novel double crosslinking IPN composites. The composites were formed taking advantage of the favourable swelling property and excellent biocompatibility of SA and the outstanding ductility and high imbibing capability of PVF. The chemical structure, microstructure, mechanical and thermal properties, water absorbing and volume expansion capacity of the IPN composites were characterized. Moreover, changes in the morphology and performance caused by the addition of different SA weight fractions were also tested. The biocompatibility of the composites referring to the effects on cell growth was investigated through cultivation of MSCs on the surface.

with a polymerization degree of 1750) was supplied by Beijing Xisi Chemicals Co. Ltd. Deionized water was used as the solvent of PVA and SA. The rest of the regents used in the experiments were all analytical reagents. 2.2. Preparation of PVF foam and the SA/PVF foamed composites The PVF foam was produced with a chemical cross-linking reaction in polyvinyl alcohol (PVA). When PVA is formalized by HCHO in the presence of an acid catalyst, the PVA is transformed into PVF. Water is removed during the reaction and the intermolecular forces cause the principal chains to aggregate, resulting in the formation of pores. The PVF formed by this process is hydrophilic and has a continuous porous structure [38]. PVA particles were dissolved into the distilled water completely with continuously stirring for 1 he1.5 h at the temperature of 90  C to prepare PVA aqueous solution. Subsequently, put the pretreated SA into PVA solution with different weight fractions to form a gel-like solution, and this mixed solution was stirred in a water bath for 0.5 he1 h at 65  C. Then the HCHO with a mass fraction of 38% was put into the mixture to get PVA formalized in the presence of an acid catalyst (sulfuric acid) and surfactant (sodium dodecyl sulfate, SDS). Then, the mixture was placed into the molds and cross-linked by an aqueous solution of 2.5% CaCl2, and rinsed with deionized water to remove excess chlorides. Finally, the composites were dried in a freeze dryer (Labconco Corporation, USA) for 12 h. The obtained composites were named as 10SA/PVF, 20SA/PVF, 30SA/PVF, 40SA/PVF and 50SA/PVF, respectively, according to the concentration of SA. 2.3. Formation of the SA/PVF composites with a crosslinking IPN SA/PVF IPN cellular materials were prepared with composition as shown in Table 1 by mechanical mixing using HCHO and CaCl2 solution as cross-linkers. PVA can be cross-linked by the formation of acetal bonds between individual PVA molecules to form a permanent network, while in the presence of calcium ions alginate Gblocks participate in gelation to form an “egg box” shaped structure network. PVA and SA formed cross-linked double networks in the presence of HCHO and calcium ions, and hence the obtained composites can be considered as IPN composites. In addition, the surface of CaCl2 drops rained on the SA were cross-linked by the alginate and covered on the PVF, and formed numbers of calcium alginate microspheres filled in the SA/PVF IPN as shown in Fig. 1. 2.4. Fourier transform infrared spectroscopy (FT-IR) The FT-IR analysis was performed on a Thermo Scientific Nicolet 6700 spectrometer. The spectra were recorded in the 4000e400 cm1 with 16 scans and a resolution of 4 cm1. 2.5. Morphological characterization by optical microscope and scanning electron microscopy (SEM)

2. Experimental

The surface images were evaluated through an optical microscope (OM) with a VHX-5000 digital microscope (Life Technologies, Eugene, OR, USA). The morphology of the sample surfaces and microstructures were further studied by FE-SEM micrographs with a FE-SEM Zeiss Supra (Apollo 300, and 10 kV). The surfaces of the samples cryogenically fractured in the liquid nitrogen were sputtered with gold to improve the image resolution.

2.1. Materials

2.6. Mechanical tests

The SA used in this study was purchased from Tianjin Guangfu Fine Chemical Research Institute. PVA powder (99% hydrolyzed,

Tensile and compression stress-strain properties were determined using a Stable Micro Systems TA-HD plus Texture Analyzer

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Table 1 Composition of SA/PVF composites. Sample

PVA (mg/mL)

SDS (g)

H2SO4 (mg/mL)

SA (mg/mL)

HCHO (mg/mL)

PVF IPN 10SA/PVF 20SA/PVF 30SA/PVF 40SA/PVF 50SA/PVF

100

0.85

19.7

0

80

100 100 100 100 100

0.85 0.85 0.85 0.85 0.85

19.7 19.7 19.7 19.7 19.7

10 20 30 40 50

80 80 80 80 80

(Stable Micro Systems Co. Ltd, UK) with the loading rate kept at a strain rate of 50 mm min1 at room temperature. The mechanical testing specimens were casted directly in the mold with the specimen dimensions following ISO standards. The porosity of the foams was calculated using the following equation [39]:

P¼

ðG2  G1 Þ=r1 ðG2  G3 þ G4 Þ=rs

where P stands for the porosity of the foams; G1 denotes the weight (N) of dry PVF or SA/PVF foams; G2 stands for the weight (N) of the samples soaked in deionized water for 24 h; G3 is the total weight (N) of the soaked foams and trays; G4 is the weight (N) of the trays; rl and rs are the densities of the pure water and the water soaking PVF or SA/PVF foams for 24 h, respectively. All the samples were measured in triplicates for each group.

Ca2þ (1%)

Immerse Immerse Immerse Immerse Immerse

2.7. Thermogravimetry (TG) and differential scanning calorimetry (DSC) Thermogravimetric experiments were carried out with a Shimadzu TGA 50 analyzer equipped with a platinum cell (Henven Corporation, Beijing). The samples went through a drying process before they were heated at a constant rate of 10  C/min from room temperature to 650  C under a N2 flow of 20 mL/min. DSC measurements were carried out in a Perkin Elmer DSC8000 (Rigaku Corporation, Japan). Samples were scanned from room temperature to 310  C at a heating rate of 10  C/min, under N2 atmosphere. Before DSC analysis, the samples were dried for 12 h. 2.8. Water content and volume expansion ratio The samples at dried state were weighted (expressed as Wd). SA/ PVF foams were immersed in distilled water at room temperature

Fig. 1. Formation of SA/PVF IPN composites.

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for 2 min to reach its equilibrium state. Then the weight (Ws) of each sample at swollen state was measured after excess surface water was removed. The equilibrium water content ratio was calculated using the following equation:

water content ¼

Ws  Wd  100% Wd

The dry specimens were cut into (10  10  0.5) mm2 for measurement, then immersed in distilled water. The volume expansivity (VE) of PVF and SA/PVF foams was determined via the following formula:

VE ¼

Vw  Vd  100% Vd

here, Vw and Vd refer to the volume of fully wetted and dry foam, respectively. All the samples were measured in triplicates for each group. 2.9. Cell culture, cell viability assay and fluorescent staining MSCs were isolated from rat bone marrow and purchased from Shanghai Institutes for Biological Sciences (Shanghai, China). The cells were cultured in primary medium containing low glucose (1.0 g/L) Dulbecco's modified Eagle's medium (L-DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillinstreptomycin (Gibco), and 4 ng/mL basic fibroblast growth factor (bFGF). Cells were kept in a 5% CO2 humidified incubator (Sanyo MCO-15AC, Japan) at 37  C. MSCs was cultured to the third generation and then used for the following experiments. Ten milligrams of the samples (PVF and SA/PVF composite) were placed in 96-well plates (Coring, USA) and exposed to 60Co irradiation (10 K) for 30 s for sterilization purpose. A CCK-8 (CCK-8, Dojindo Molecular Technology, Japan) was used to quantitatively evaluate cell viability on PVF and SA/PVF composites after cultivation for 1, 3, and 7 days. MSCs were seeded on the samples at a density of 3  104 cells per cm2, while cells cultured on the 96well plates at the same density were used as the control group. After certain times, the samples were moved to a new 96-well plate filled with 180 mL of serum-free L-DMEM medium plus 20 mL of CCK-8 solution per well. After a 6 h incubation at 37  C, the resultant production of water-soluble formazan dye was assayed at a wavelength of 450 nm by a microplate reader (MULTISKAN MK3, Thermo, USA). Three parallel replicates for each sample were used. To investigate the cell adhesion on PVF and SA/PVF composites, the MSCs on the samples were stained with F-actin after 72 h of normal culture. Briefly, the cells were washed with 1  PBS, fixed with 4% paraformaldehyde solution in PBS for 20 min, treated with 0.1% Triton X-100 (Sigma, USA) for 5 min, and blocked with 1% bovine serum albumin (Sigma, USA) for 60 min. The cellular actin filaments were then stained with phalloidin conjugated to AlexaFluor 488 (Invitrogen, USA) at a 1:200 dilution for 120 min, and the nuclei were stained with 40 , 6-diamidino-2-phenylindole (DAPI, 300 nM, Life Technology, Switzerland) for 10 min. The cells were washed three times with 1  PBS (Phosphate Buffer Saline) after each step. Then the samples were observed under a fluorescence microscopy (Life Technologies, Eugene, OR, USA). 2.10. Statistical analysis One-way analysis of variance (ANOVA) was carried out using Statistical Product and Service Solutions (SPSS) software to detect the statistical significance of the collected data. All the data were

expressed as means ± standard deviation (SD). *p < 0.05 represents significant difference among mean values. 3. Results and discussion 3.1. Chemical structure characterization The FT-IR spectra of primordial PVF and the SA/PVF composites are shown in Fig. 2. The five absorption bands between 1000 and 1250 cm1 were the characteristic peaks of polyvinyl formal, which was generated by the contraction and vibration of the acetal bond with the structure of 1, 3 Dioxane [40]. As it is shown in the FT-IR spectra, all the specimens had the acetal bond except SA. The large band observed between 3600 and 3200 cm1 was linked to the stretching of OeH, including the hydroxyl groups and the intramolecular and intermolecular hydrogen bonds between PVF and SA. With the increasing proportion of SA, this band became broader and was shifted to lower wavenumbers. This could be due to the increased amount of hydroxyl groups in the samples. The absorption band between 2840 and 2950 cm1 referred to the asymmetric and symmetric stretching modes ofeCH2 from alkyl groups, which was shifted to lower wavenumber and its intensity decreased with the addition of SA. Compared with the FT-IR curve of the original PVF, new absorption peak of SA/PVF appeared at 1736 cm1 and was assigned to the C]O stretching vibration, which was the characteristic peak of SA. This indicated that SA was successfully added into PVF and SA/PVF composites were formed. The intensity of the C]O stretching vibrations increased with the addition of SA, therefore the flexibility of the SA/PVF samples could be better [41]. The band observed at 1631 cm1 corresponds to the absorbed water or the results of d (HOH) [42]. It shifted to higher wavenumber with higher SA ratio in foamed composites, because of the stronger molecular interactions between PVF and SA. Also, there was an obvious difference around 1020 cm1 among all the SA/PVF samples. The stretching vibration intensity of CeOeC got lower with the increasing SA content. This manifested that SA content had a significant influence on polymer structure. Besides, as shown in the FT-IR spectra, the intensity of the acetal bond group obviously decreased with the addition of SA. This indicated that introducing SA could brought in a number of hydrophilic hydroxyl, thereby weakening the infrared absorption of the acetal groups and improving the softness of composites. 3.2. Morphological characterization In general, the physical properties of foams depend not only on the rigidity and hydrophilia of the polymer matrix, but also on the cell structures. Thus, it is significant to observe the structure of the foamed specimens using OM. The OM images and pore size distribution of PVF and SA/PVF foams with different SA content are shown in Fig. 3. It can be seen that all the foamed composites possessed roughly open cell structures and the foams displayed an interconnecting, porous network and a spongy-like structure. As shown in Fig. 3 and 50SA/PVF had the smallest average cell size (87.33 ± 4.83 mm) in comparison with PVF (202.08 ± 10.06 mm), 10SA/PVF (151.64 ± 7.37 mm), 20SA/PVF (143.92 ± 6.58 mm), 30SA/ PVF (135.82 ± 4.85 mm) and 40SA/PVF (96.53 ± 4.94 mm). PVF presented a non-uniform cell size distribution and the pores were large, irregular and uneven. With the increasing SA concentration, the pore size decreased and the uniformity of pore size distribution increased. Among all the foams, the 30SA/PVF composite had the most uniform porosity distribution. It is reported that the pore sizes of PVF foams are generally larger than 25 mm, and the average pore diameter of various modified PVF varies from 10 to 100 mm [43]. In general, it is difficult to obtain modified PVF with large and uniform

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Fig. 2. FT-IR spectra of primordial PVF and the SA/PVF composites.

pores. However, through adding SA, the SA/PVF composite formed a favorable cellular structure, and the pore size of it was relatively regular and varied from 87 to 150 mm. Besides, PVF modified by different content of SA could form porous materials with different pore size and different mechanical properties, so as to be multifunctional in the biomedical field. Fig. 4 shows the SEM images of the PVF, 10SA/PVF, 20SA/PVF, 30SA/PVF, 40SA/PVF, and 50SA/PVF composites. It was obvious that the PVF net structure became thicker and tended to gather into lamellar structure with additive SA. The SEM micrographs showed composites with a three-dimensional interconnection structure throughout. The pore size of the composites fell in the range 20e200 mm, which was suitable for use in wound healing [44]. With the addition of HCHO and Ca2þ, SA/PVF composites with double cross-linking IPN structures were obtained. In addition, the SA/PVF composites had a unique structure consisting of a top skin layer and a sponge-like porous layer. The existence of the cellular structure provided great probability for applications in tissue engineering and wound dressings [45]. 3.3. Physical and mechanical properties The PVF and (10, 20, 30, 40 and 50) SA/PVF composites were subject to both compressive and tensile tests. Typical stressestrain curves of the samples are depicted in Fig. 5. The changes in the compressive and tensile properties of the composites induced by the addition of SA and the data of the physical and mechanical properties of all the materials are summarized in Table 2. Fig. 5(a) shows the tensile stressestrain graphs of the PVF and SA/PVF composites. As high elastic materials, the samples were broken in the linear elastic region without obvious yield phenomenon and further plastic deformation. For 10SA/PVF, 20SA/PVF, 30SA/PVF, 40SA/PVF and 50SA/PVF, the tensile strengths were 288%, 326%, 462%, 417% and 221% of the original values for PVF, respectively (Table 2). It can be seen that the modulus of composites was higher than that of PVF. With the increase of SA content, the modulus raised dramatically and then declined. The highest modulus was 2.348 KPa for 30SA/PVF, and the highest strength

reached 614 KPa. The modulus increased 1857% compared to the original PVF, and the strength increased 361%, simultaneously. The elongation of 30SA/PVF composites declined slightly to about 470% (600% for PVF). When weight ratio of SA was over 50%, the elongation decreased dramatically to around 300%. The reduction in the tensile modulus and the elongation from 30SA/PVF to 50SA/PVF might attribute to the better SA dispersion in the former, which was favorable for load transfer. With the increasing addition of SA, the hydrogen bonds and the intermolecular interaction between SA and PVF reached saturation. Excess SA tangled with PVF molecular chains, and formed large pieces of layers over PVF chains, meanwhile reduced the number of pores as well as the strength and modulus of the composites. Fig. 5(b) shows the compression stress-strain curves of SA/PVF composites with different contents of SA. As shown in Fig. 5(b), the compressive stressestrain curves showed linear elasticity at low strains, followed by a plateau-like region, and approached a cellular solid structure [46]. After loading force, the orientations and relative positions of the blend polymers chains were thought to be changed, and then the interstitial liquid was squeezed out. Thus, the stress increased with the increasing strain at the initial stage. During the compression process, the chain orientations tended to be uniform, and the frictional drag force increased. As a result, it would be more difficult to compress at high strain [47]. For the cellular materials, when the pores almost completely collapsed, the opposing cell walls were in contact, and further strain compressed the solid itself. This was the densification region in which the stress rose steeply with strain. The compression modulus was calculated and shown in Table 2. It can be seen that with the increasing addition of SA, the compression modulus of the composites increased dramatically at first, but then decreased significantly when the weight ratio of SA was over 30%. The improved compression property attributed to good dispersion of SA in composites and the interaction of the hydrogen bond between hydrophilic SA and PVF, which was conductive to transfer and share the stress. But when the SA content increased to over 30%, free hydroxyl and crosslinking points tended to be saturated, meanwhile nonuniformly distributed SA contributed to more defects rather than

14 Y. Wang et al. / Composites Part B 121 (2017) 9e22

Fig. 3. Microscope images and the pore size distributions of PVF and SA/PVF composites with different SA content.

Y. Wang et al. / Composites Part B 121 (2017) 9e22

Fig. 4. SEM micrographs of PVF and SA/PVF foams with different SA weight fractions: 10SA/PVF, 20SA/PVF, 30SA/PVF, 40SA/PVF and 50SA/PVF.

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Fig. 5. Effect of SA concentration on the mechanical properties of SA/PVF foams: (a). Tensile stress-strain curves (b). Compression stress-strain curves (c). Compressive strength and compressive resilience (d). Porosity. SA, refers to alginate hydrogel cross-linked by calcium ions.

Table 2 Mechanical Properties of the SA/PVF composites. Sample

Tensile strength (KPa)

PVF 10SA/PVF 20SA/PVF 30SA/PVF 40SA/PVF 50SA/PVF

133.31 382.87 433.77 614.34 554.52 294.99

± ± ± ± ± ±

3.42 10.17 9.98 16.11 15.37 6.22

Modulus (KPa) 0.1201 0.8879 1.3504 2.3477 2.0575 0.9450

± ± ± ± ± ±

0.0055 0.0418 0.0699 0.1769 0.0957 0.0482

Elongation at break (%) 609.54 491.68 448.91 428.28 368.13 320.24

± ± ± ± ± ±

reinforcement in the composites, thus causing the compression properties to decline sharply. Fig. 5(c) shows the compressive strength and compressive resilience of SA/PVF composites with different content of SA. As shown in Fig. 5(c), the compressive resilience of these samples were all above 95%, and they could all be close to full recovery. However, the compressive strength increased significantly except the pristine PVF. That means the SA/PVF composites could still have

21.32 14.84 12.56 9.86 12.51 8.96

Compressive strength (KPa)

Compressive resilience (%)

12.91 ± 0.32 26.88 ± 0.56 78.32 ± 2.19 155.37 ± 4.65 131.86 ± 3.55 96.29 ± 2.12

99.52 98.12 95.25 96.87 95.17 95.63

± ± ± ± ± ±

1.07 1.09 1.42 1.16 1.62 1.33

good resilience at high compression strength. The porosity of the composites is shown in Fig. 5(d). It indicates that the porosity decreased with the increasing SA content. As shown in SEM images, with increasing SA content, SA tended to gather into lamellas and coated on the chains of PVF after crosslinked by Ca2þ, thus, the pore size and porosity of the composites decreased significantly. The strength of the porous material was gradually enhanced with the increasing of the relative density.

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With the decreasing of porosity, the relative density increased along with the breaking stress, and the tensile deformation of the porous materials became more difficult, so that the elongation at break decreased. However, the mechanical properties of the composites improved dramatically and reached optimal value at 30SA/PVF. We speculate that in the 30SA/PVF sample, the SA dispersed most uniformly. Moreover, the 30SA/PVF IPN foamed composite has the

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highest tension of 0.6 MPa and elongation of 450%, so tough that it could withstand further handling and post processing. 3.4. Thermal behavior analysis The corresponding values of thermogravimetric curves are shown in Table 3. The TG and DTG analyses of SA, PVF, 20SA/PVF, 30SA/PVF, and 40SA/PVF are illustrated in Fig. 6 (a) and (b),

Table 3 The parameters of TG and DTG of SA, PVF, and SA/PVF foams with different SA content. Sample

Onsite ( C)

Tdmax ( C)

Rmax (% min1)

Carbon residue rate (%)

SA PVF 20SA/PVF 30SA/PVF 40SA/PVF

180.7/275.4 355.4 337.2 316.6 302.5

197.2/393.7 403.1 339.3 401.6 400.3

3.22/-4.41 18.85 17.50 12.92 12.57

58.34 10.60 19.53 23.69 24.01

Fig. 6. (a) TG, (b) derivative TG curves and (c) DSC thermograms for the SA, PVF, and SA/PVF composites.

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respectively. The onset degradation temperatures of SA, PVF, 20SA/ PVF, 30SA/PVF, 40SA/PVF, were 190  C, 380  C, 360  C, 345  C and 330  C, respectively. It can be seen that the three SA/PVF IPN foams had similar behavior under a nitrogen atmosphere. The composites showed lower onset degradation temperatures than the PVF; the SA deposited on the PVF surface started to decompose at higher temperatures (increasing to 140, 155, and 170  C) compared with pure SA. Besides, with the increase of SA content, the maximum degradation temperature remained stable (Fig. 6(b)). Both phenomenon could be explained by the excellent compatibility between the two components of the composites. In addition, in the TG curves, except for PVF, all the samples presented a residual mass of about 20% or more. This may attribute to the SA layers coating on the chains of PVF, which delayed the thermal decomposition process. DSC results for the PVF and the SA/PVF foamed composites with different content of SA are shown in Fig. 6(c). The SA/PVF composites had two distinct glass transition temperatures (Tg), which represented the characteristic Tg of SA and PVF respectively. In consistent with the preceding analysis of FT-IR and SEM, it indirectly illustrated that S A blended with PVF and formed compatible and stable IPN composites [48]. Because of the higher crosslinking degree, the Tg of SA/PVF composites are higher than that of PVF. The

increased entanglements of SA/PVF chains also contributed to higher Tg for the SA/PVF composites. With the increasing of SA content, the Tg of SA/PVF samples increased. This phenomenon resulted from the interaction between SA and PVF. SA layers adhered with the PVF chains, which were likely to inhibit glass transition by restricting the mobility of surrounding PVF chains and improving their thermal stability. 3.5. The water absorbing capacities and volume expansion rate To behave as superabsorbent polymers (SAP), the water absorption capacity of porous materials is one of the most important properties needed to be identified [49], therefore we have tested the water absorption capacity and the results are shown in Fig. 7(aed). It was found that the water absorptions of both PVF and SA/PVF were well above that of SA (cross-linked by Ca2þ). The water content of 10SA/PVF, 20SA/PVF and 30SA/PVF was almost the same with that of the primitive PVF. But the water content of 40SA/PVF and 50SA/PVF showed a slight decrease. Moreover, the water absorption rate gradually reduced with higher mass content of SA. It may be attributed to the increase of crosslinking points, crosslinking degree, and the formation of more compact IPN network, so that the micro pores became smaller and the net structure became

Fig. 7. Water absorbing capacities of the SA/PVF composites: (a, b & c) water absorption rate and (d) water content.

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tighter, thereby hindering the water entering the composites. The volume expansion rate and starting expansion time of PVF and the SA/PVF composites with different content of SA are shown in Fig. 8. For 10SA/PVF, 20SA/PVF, 30SA/PVF, 40SA/PVF and 50SA/ PVF, the volume expansion rate were 1550%, 2020%, 2150%, 1750% and 900%, respectively. The hydroexpansivity of SA/PVF composites were significantly better than that of pristine PVF (1200%) and other modified PVF (1000%e1400%) [15]. Movie S1, Movie S2, and Fig. 8(cef) show the volume expansion rate of PVF and SA/PVF. It can be visually seen from the figure that, the SA/PVF composites had excellent expansion capabili ties, which were induced by the combined action of water absorption expansion of PVF and the swelling of SA. Besides, blending with alginate could disrupt the hydrogen bonding between the PVF, which was another possible reason for the increase in the expansion ability. Fig. 8(a) also indicated that the composites (except 50SA/PVF) had excellent expansion capacities (at least 15.5 times of its dry size) after adding SA. However, it may lower down the expansion rate with more SA (over 30%). When the SA content increased to over 30%, free hydroxyl and crosslinking points tended to be saturated, meanwhile non-uniformly distributed SA contributed to more defects. Besides, excess SA tangled with PVF molecular chains, and formed large pieces of layers over the IPNs. This, however, would increase the relative densities and reduce the

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number of pores in the composites. The increase of relative density would be adverse for the water swelling of the proposed composites. The decrease of the internal pores might be difficult for water molecules to enter the composites. With the increasing of SA content, the relative density of 40SA/PVF and 50SA/PVF successively increased, and the amount of internal pores decreased sharply, which caused the rapid decline of volume expansion rate, as shown in Fig. 8. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.compositesb.2017.01.045. SA/PVF composites have excellent rapid-imbibing capacity and unique high-rate hydro-expansive property, thus it can be applied to surgery to clean exudate and stop bleeding. It could also be used as filling sponges and play a supporting role in hemostasis. Furthermore, SA/PVF composites with high expandable ratio and nearly 100% recoverable strain will be potentially used as shape memory polymers (SMPs) [50,51]. 3.6. Cell proliferation and cell morphology MSCs have high growth rate, ability to self-renewal and potential of multiple differentiation. Therefore, MSCs have been frequently used as a model to study the cytocompatibility of different samples, including cell adhesion, spreading and

Fig. 8. volume expansion rate (a) and starting expansion time (b) of PVF and SA/PVF composites. Pictures of SA/PVF before (c) and after (d) absorbing water, PVF before (e) and after (f) absorbing water.

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proliferation[52, 53]. The biocompatibilities of PVF and 30SA/PVF were evaluated after culturing with MSCs for several days. CCK-8 assays were performed to assess the proliferation of the cells incubated on samples in this study and the results are shown in Fig. 9. With the culturing time increasing from 1 day to 7 days, the viability of cells on each sample showed a significant increase. The cell number increased rapidly on both foams after 3 days, and the cells on the SA/PVF composite had a higher proliferation rate than

Fig. 9. Proliferation of MSCs cultured on PVF and SA/PVF composite in 7 days was measured by a CCK-8 assay *p < 0.05 compared to the control.

on the PVF sample. Besides, no significant difference could be observed between SA/PVF and the control group. After 7 days, the cell number on the SA/PVF composite was about ~1.14- fold higher than that on the PVF. It is mainly due to that SA is a natural polysaccharide, which is conductive to the cell adhesion. Furthermore, the relative cells number on the SA/PVF composite was notably increased compared to the control group. The results suggest that the SA/PVF composite has excellent cytocompatibility, and the addition of SA in PVF makes it better for MSC proliferation. As shown in Fig. 10, after culturing for 1 day, it can be seen that MSCs were sparsely spread everywhere with tiny dense cell domains. MSCs on all samples exhibited polygonal morphology, and there were no significant differences on the cell numbers between the sample and the control group. On the 5th day, a large amount of MSCs were adherent on both the SA/PVF sample and the control group, evenly distributed and formed a monolayer. However, there were fewer cells on the PVF sample, revealing its poor biocompatibility performance. Cell morphology was observed by fluorescence microscope after staining with phalloidin and DAPI. The MSCs were cultured on different samples for 7 days. As shown in Fig. 11, the first row shows the Actin-stained intact cytoskeleton of MSCs and the second row shows the DAPI-stained intact nuclei of MSCs. The cells that covered on the SA/PVF composites with a typical geometry structure had an obvious better cell adhesion status than that cultured on the PVF, and the cells aggregated and formed clusters on the composite, which indicated that the SA/PVF composite with porous morphology was suitable for the adhesion of MSCs. The fluorescence images also suggest that MSCs spread uniformly around the SA/PVF composite. Additionally, from the quantity of nuclei (DAPI,

Fig. 10. OM images of MSCs on PVF and SA/PVF after culturing for 1 d and 5 d.

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Fig. 11. The morphology of MSCs incubated on three different samples. After 7 days, cells were fixed and subjected to phalloidin (green) and DAPI staining (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

blue), the amount of cells on the SA/PVF composite was much more noticeable than that on the PVF. After culturing for 7 days, large numbers of cells were relatively evenly distributed on the SA/PVF sample. The number of cells on the PVF sample was significantly lower compared with the control group, and the SA/PVF sample presented the largest number of cells. All the results show that the cell adhesion and proliferation are better on the SA/PVF composite than that on the PVF and control samples. 4. Conclusions A novel foamed composite with controllable three-dimensional IPN structure and surface morphology was prepared through the blending of PVA with SA, followed by cross-linking with formaldehyde and Ca2þ. The chemical structure, micromorphology, mechanical and thermal properties, water absorbing and volume expansion capacity of SA/PVF composites changed with the different content and distribution of SA. The uniform interconnected pores endow the SA/PVF foams with unique spongy morphology and good resilience, both of which are controlled by the addition of SA. The physical and mechanical properties of PVF were also significantly enhanced with the addition of SA. Besides, the SA/PVF composites showed excellent expansion capabilities, the highest expansivity of which reached 2150%. The changes in these properties were attributed to the formation of the

crosslinking IPN, which showed a compact ionic crosslinking surface and a tough, cellular structure. The formation of this special structure dramatically improved the thermal and mechanical properties as well as hydroexpansivity of the composites. Furthermore, SA/PVF samples enhanced the cell proliferation of MSCs, and the cells incubated on the surfaces showed good morphology after a period of time. The results showed that the prepared IPN composites may be applied in the medical sponges, tissue engineering scaffolds, wound dressing and surgical filling sponges because of the combined outstanding solution absorbing performance, excellent mechanical strength, unique high-rate hydroexpansivity and promising biocompatibility. Acknowledgement This study is financially supported by National Natural Science Foundation of China (Grant No. 51273021 and 51473019) and Beijing Municipal Science and Technology Plan Projects (No. Z161100000116003). References [1] Rinaudo M. Chitin and chitosan: properties and applications. Prog Polym Sci 2006;31(7):603e32. [2] Ramli NA, Wong TW. Sodium carboxymethylcellulose scaffolds and their physicochemical effects on partial thickness wound healing. Int J Pharm

22

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2011;403(1):73e82. [3] Mi F-L, Shyu S-S, Wu Y-B, Lee S-T, Shyong J-Y, Huang R-N. Fabrication and characterization of a sponge-like asymmetric chitosan membrane as a wound dressing. Biomaterials 2001;22(2):165e73. [4] Chiaoprakobkij N, Sanchavanakit N, Subbalekha K, Pavasant P, Phisalaphong M. Characterization and biocompatibility of bacterial cellulose/ alginate composite sponges with human keratinocytes and gingival fibroblasts. Carbohydr Polym 2011;85(3):548e53. [5] Buzarovska A, Gualandi C, Parrilli A, Scandola M. Effect of TiO 2 nanoparticle loading on poly( l -lactic acid) porous scaffolds fabricated by TIPS. Compos Part B Eng 2015;81:189e95. [6] Xu K, Li K, Zhong T, Guan L, Xie C, Li S. Effects of chitosan as biopolymer coupling agent on the thermal and rheological properties of polyvinyl chloride/wood flour composites. Compos Part B Eng 2014;58(3):392e9. [7] Hou B-s, Zheng Y-d, Yue L, Wang C, Qiao K, Cui L-y, et al. Effects of degree of acetalization and pore structure on water absorbency and mechanical properties of polyvinyl formal (PVF) porous material. Acta Polym Sin 2015;(6): 657e66. [8] Qiao K, Zheng Y, Guo S, Tan J, Chen X, Li J, et al. Hydrophilic nanofiber of bacterial cellulose guided the changes in the micro-structure and mechanical properties of nf-BC/PVA composites hydrogels. Compos Sci Technol 2015;118: 47e54. [9] Yu Q, Zheng Y, Yan N, Xie Y, Qiao K, Jin R. The gelation process and protein absorption property of injectable SA-CMBC hydrogel used for procoagulant material. Rsc Adv 2015;5(129):106953e8. [10] Pan Y, Shi K, Peng C, Wang W, Liu Z, Ji X. Evaluation of hydrophobic polyvinylalcohol formaldehyde sponges as absorbents for oil spill. ACS Appl Mater Interfaces 2014;6(11):8651e9. [11] Smith L, Ma P. Nano-fibrous scaffolds for tissue engineering. Colloids Surfaces B Biointerfaces 2004;39(3):125e31. [12] Ma Z, Kotaki M, Inai R, Ramakrishna S. Potential of nanofiber matrix as tissueengineering scaffolds. Tissue Eng 2005;11(1e2):101e9. [13] Lian F, Guan H-y, Wen Y, Pan X-r. Polyvinyl formal based single-ion conductor membranes as polymer electrolytes for lithium ion batteries. J Membr Sci 2014;469:67e72. [14] Guan HY, Guo ZB, Ding JJ, Lian F. Ionic conductivity and dielectric behavior of novel poly(vinyl formal)-based single-ion-conductor polymer electrolytes, vol.133; 2016 (23):[n/a-n/a]. [15] Pan Y, Wang W, Peng C, Shi K, Luo Y, Ji X. Novel hydrophobic polyvinyl alcoholeformaldehyde foams for organic solvents absorption and effective separation. RSC Adv 2014;4(2):660e9. [16] Xue B, Li R, Deng J, Zhang J. Sound absorption properties of microporous poly(vinyl formal) foams prepared by a two-step acetalization method. Industrial Eng Chem Res 2016;55(14):3982e9. [17] Fitzhugh AF, Lavin E, Morrison GO. The manufacture, properties, and uses of polyvinyl formal. Mater Lett 2012;72(8):57e9. [18] Takashima T, Hoshino R, Nakanishi Y, Higaki H. An application of polyvinyl formal to total joint replacement. Berlin Heidelberg: Springer; 2007. [19] Togami W, Sei A, Okada T, Taniwaki T, Fujimoto T, Nakamura T, et al. Effects of water-holding capability of the PVF sponge on the adhesion and differentiation of rat bone marrow stem cell culture. J Biomed Mater Res Part A 2014;102(1):247e53. [20] Miyoshi H, Ookawa K, Ohshima N. Hepatocyte culture utilizing porous polyvinyl formal resin maintains long-term stable albumin secretion activity. J Biomaterials Sci Polym Ed 1998;9(3):227e37. [21] Huang J, Yang L, Wang X, Li H, Chen L, Liu Y-N. A novel post-cross-linked polystyrene/polyacryldiethylenetriamine (PST_pc/PADETA) interpenetrating polymer networks (IPNs) and its adsorption towards salicylic acid from aqueous solutions. Chem Eng J 2014;248:216e22. [22] Karadaǧ E, Sh Kundakci. Water and dye uptake studies of acrylamide/4styrenesulfonic acid sodium salt copolymers and semi-interpenetrating polymer networks composed of gelatin and/or PVA. Adv Polym Technol 2013;32(S1):E531e44. [23] Klempner D, Sperling L, Utracki L. Advances in chemistry. Washington, DC: American Chemical Society; 1994. [24] Girija P, Beena M. Sorption of trace amounts of Pb (II) ions on an ion imprinted interpenetrating polymer network based on alginic acid and crosslinked polyacrylamide. Sep Sci Technol 2014;49(7):1053e61. [25] Li Q, Liu J, Su Y, Yue Q, Gao B. Synthesis and swelling behaviors of semi-IPNs superabsorbent resin based on chicken feather protein. J Appl Polym Sci 2014;(1):131. [26] Liao W, Gao S, Xie X, Xu M. Macroporous crosslinked hydrophobic/hydrophilic

[27]

[28]

[29] [30]

[31] [32]

[33] [34] [35]

[36]

[37]

[38] [39]

[40] [41] [42]

[43]

[44] [45] [46] [47]

[48] [49] [50] [51] [52]

[53]

polystyrene/polyamide interpenetrating polymer network: synthesis, characterization, and adsorption behaviors for quercetin from aqueous solution. J Appl Polym Sci 2010;118(6):3643e8.
che B, Panouille  M, Larreta-Garde V. Gelatin-polysaccharide Picard J, Doume mixed biogels: enzyme-catalyzed dynamics and IPNs. macromolecular symposia. Wiley Online Library; 2010. p. 337e44. Brigham MD, Bick A, Lo E, Bendali A, Burdick JA, Khademhosseini A. Mechanically robust and bioadhesive collagen and photocrosslinkable hyaluronic acid semi-interpenetrating networks. Tissue Eng Part A 2008;15(7):1645e53. Suri S, Schmidt CE. Photopatterned collagenehyaluronic acid interpenetrating polymer network hydrogels. Acta biomater 2009;5(7):2385e97. Kuila SB, Ray SK. Dehydration of dioxane by pervaporation using filled blend membranes of polyvinyl alcohol and sodium alginate. Carbohydr Polym 2014;101:1154e65. Stone SA, Gosavi P, Athauda TJ, Ozer RR. In situ citric acid crosslinking of alginate/polyvinyl alcohol electrospun nanofibers. Mater Lett 2013;112:32e5. Liling G, Di Z, Jiachao X, Xin G, Xiaoting F, Qing Z. Effects of ionic crosslinking on physical and mechanical properties of alginate mulching films. Carbohydr Polym 2016;136:259e65. Gilchrist T, Martin A. Wound treatment with sorbsandan alginate fibre dressing. Biomaterials 1983;4(4):317e20. Fraser R, Gilchrist T. Sorbsan calcium alginate fibre dressings in footcare. Biomaterials 1983;4(3):222e4. Suzuki Y, Tanihara M, Nishimura Y, Suzuki K, Yamawaki Y, Kudo H, et al. In vivo evaluation of a novel alginate dressing. J Biomed Mater Res 1999;48(4):522e7. Lee J, Bhang SH, Park H, Kim B-S, Lee KY. Active blood vessel formation in the ischemic hindlimb mouse model using a microsphere/hydrogel combination system. Pharm Res 2010;27(5):767e74. Kolambkar YM, Dupont KM, Boerckel JD, Huebsch N, Mooney DJ, Hutmacher DW, et al. An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects. Biomaterials 2011;32(1):65e74. Flory PJ. Statistical mechanics of reversible reactions of neighboring pairs of substituents in linear polymers. J Am Chem Soc 1950;72(11):5052e5. Gong R, Xu Q, Chu Y, Gu X, Ma J, Li R. A simple preparation method and characterization of epoxy reinforced microporous phenolic open-cell sound absorbent foam. RSC Adv 2015;5(83):68003e13. Stuart B. Infrared spectroscopy. Wiley Online Library; 2005. Brinson HF, Brinson LC. Polymer engineering science and viscoelasticity. Springer; 2008.  Caki Miti c Z, c M, Nikoli c GM, Nikoli c R, Nikoli c GS, Pavlovi c R, et al. Synthesis, physicochemical and spectroscopic characterization of copper (II)-polysaccharide pullulan complexes by UVevis, ATR-FTIR, and EPR. Carbohydr Res 2011;346(3):434e41. Artyukhov A, Shtilman M, Kuskov A, Pashkova L, Tsatsakis A, Rizos A. Polyvinyl alcohol cross-linked macroporous polymeric hydrogels: structure formation and regularity investigation. J Non-Crystalline Solids 2011;357(2): 700e6. Rovee DT. Evolution of wound dressings and their effects on the healing process. Clin Mater 1991;8(3):183e8. Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 2005;26(27):5474e91. Gibson LJ, Ashby MF. Cellular solids: structure and properties. Cambridge university press; 1999. ~ a LN, Ponce A, Alvarez VA. Poly (vinyl alcohol)/cellulose Gonzalez JS, Luduen nanowhiskers nanocomposite hydrogels for potential wound dressings. Mater Sci Eng C 2014;34:54e61. Wang J, Ye L. Structure and properties of polyvinyl alcohol/polyurethane blends. Compos Part B Eng 2015;69(69):389e96. Buchholz FL, Graham AT. Modern superabsorbent polymer technology, 279. 605 Third Ave, New York, NY 10016, USA: John! Wiley & Sons, Inc; 1998. Langer R, Tirrell DA, Langer R, Tirrell DA. Designing materials for biology and medicine. Nature 2004;428(6982):487e92. Zhang L, Du H, Liu L, Liu Y, Leng J. Analysis and design of smart mandrels using shape memory polymers. Compos Part B Eng 2014;59(59):230e7. €se S, Korkusuz P, Timuçin M, Korkusuz F. Boron containing nano Ciftci E, Ko hydroxyapatites (B-n-HAp) stimulate mesenchymal stem cell adhesion, proliferation and differentiation. Key Eng Mater 2014;631(3):373e8. Ragetly GR, Griffon DJ, Lee HB, Yong SC. Effect of collagen II coating on mesenchymal stem cell adhesion on chitosan and on reacetylated chitosan fibrous scaffolds. J Mater Sci Mater Med 2010;21(8):2479e90.