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Development of Nanohybrid Interpenetrating Network (IPN) Hydrogel of Laponite

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The aim of this study was to develop nanohybrid interpenetrating network (IPN) hydrogel of laponite: polyvinyl alcohol (PVA)-alginate with adjustable mechanical and degradation properties and potential hemostatic characteristic for wound healing and tissue regeneration application. In this study, after solution mixing of alginate (4 wt.%) and PVA (10 wt.%) with various concentrations of laponite (0, 0.5, 1, and, 2 wt.%), IPN hydrogel was prepared via subsequently ionically and covalently crosslinking of alginate and PVA, respectively. The chemical and structural properties of the nanohybrid hydrogels were evaluated using Furrier transform Infrared spectroscopy, X-ray diffraction, scanning electron microscopy and transmission electron microscopy. Moreover, the effect of laponite concentration on the degradation rate, water swelling ability and mechanical properties of the nanohybrids were estimated. Result demonstrated that compared to PVA-alginate IPN film, mechanical strength of Laponite: PVA-Alginate significantly enhanced (upon 2 times) which might be due to  the additional crosslinking of PVA-Alginate chains with laponite nanoplatelets through noncovalent interactions. Moreover, incorporation of 2 wt.% laponite reduced swelling ability (3 times) and degradation ratio (1.2 times) originating from effective enhancement of crosslinking density in nanohybrid hydrogels which could retard water absorption. Furthermore, as prepared nanohybrid hydrogels revealed admirable biocompatibility against MG63 and human fibroblast skin cells. Noticeably, the MTT assay demonstrated that the proliferation of human fibroblast skin cells significantly enhanced on hybrid hydrogel containing 0.5 wt.% laponite compared to tissue culture plate (control) (1.5 folds) and PVA-alginate hydrogel (1.4 folds). In addition, hemolysis and clotting test indicated that the nanohybrid hydrogels could promote hemostasis which could be helpful in wound dressing. Therefore, the synergistic effects of nanohybrid hydrogel such as superior mechanical properties, adjustable swelling properties and degradation rate as well as admirable biocompatibility and hemolysis encouraging coagulation ability in a hemostat makes it a desirable candidate for treating incompressible wounds in emergency conditions.

  1. Introduction

Hydrogels as extremely hydrophilic macromolecular networks are attractive candidates for a range of biomedical applications due to their physical and chemical properties often mimicking the extracellular matrix (ECM) of native soft tissue (Lee & Mooney, 2001). Between various applications, hydrogels have been particularly attractive in wound healing process. The principal goal of wound healing is to prompt recovery with negligible scarring and maximal function. In this way, essential characteristics of membranes as wound dressings to support the skin healing and keep the skin deficiency zone from contamination have been increasingly considered. As for wound healing process, hydrogel dressing could preserve a humid environment around the wound and retain the wound exudates promoting fibroblast proliferation and keratinocyte migration (Jhong et al., 2014). Furthermore, formation of clot is one of the crucial process for wound healing which establish hemostasis by concentrating clotting factors and forming a physical barrier against bleeding, to the injured site to promote clotting (Gaharwar et al., 2014). However, the applications of hydrogels have often be limited due to their inadequate mechanical strength and toughness as well as high degradation rate (Annabi et al., 2014). Conventionally, hydrogels typically exhibit low elastic modulus, tensile and compressive strength, while retaining high swelling ratios (10 – 100) and degradation rate (Costa & Mano, 2015).
Numerous strategies have proposed to enterprise hydrogels with superior mechanical properties.  Basically, hydrogels with excellent mechanical characteristics could be categorized in three main types consisting of topological (TP), nanocomposite (NC) and double network (DN) gels (Haraguchi & Takehisa, 2002; Tanaka, Gong, & Osada, 2005). DN gels comprise of two interpenetrating polymer networks (IPN); rigid and flexible polymers. DN gels are 3D networks comprising of two or more polymeric networks, partly or entirely intertwined on a molecular scale, while not covalently joined to each other and could not be separated except once chemical bonds are destroyed (Hoare & Kohane, 2008). Results reveal that IPN hydrogels afford both mechanical strength and toughness when one component is cross-linked covalently and other one is ionically cross-linked (Naficy, Kawakami, Sadegholvaad, Wakisaka, & Spinks, 2013). Recently, various hybrid ionic-covalent IPN hydrogels have been developed consisting of polyacrylamide (PAAm)- alginate (Darnell et al., 2013), poly(acrylic acid) (PAA)-alginate (Lin, Ling, & Lin, 2009) and polyvinyl alcohol (PVA)-alginate (Golafshan, Kharaziha, & Fathi, 2016; Thankam, Muthu, Sankar, & Gopal, 2013). Due to the cytocompatibility, hydrophilicity, water solubility, suitable mechanical properties as well as low price, PVA has been widely applied for engineering various tissues such as bone (Nie et al., 2012), heart (Thankam et al., 2013), nerve (Golafshan et al., 2016)  and vascular network (Thomas, Arun, Remya, & Nair, 2009). Moreover, alginate is a negatively charged polysaccharide derived from brown seaweed, which has been widely applied to develop IPN hydrogels. The negative charge of alginate originates from the carboxyl groups sited on the ring structure of both b-D mannuronate (M block) and a-L guluronate (G) monomers (K. Shalumon et al., 2011). Due to non-toxicity, biodegradability and biocompatibility, alginate has been extensively employed for tissue engineering application (Wong, 2004) and cell therapy (Orive, Tam, Pedraz, & Hallé, 2006). Recently, PVA-Alginate fibrous scaffold was developed using electrospinning followed by two-step cross-linking process. Despite the significant mechanical properties of PVA-alginate compared to pure PVA and alginate, degradation rate and hemostasis properties due to the hydrophilic nature of polymers are not appropriate for skin tissue regeneration. Furthermore, mechanical properties, specifically toughness, were not satisfied (Golafshan et al., 2016).
Recently, nanohybrid hydrogels consisting of various kinds of nano-fillers such as graphene, hydroxyapatite, carbon nanotubes, metallic nanoparticles have gained importance due to their properties which could be easily modulated (Kharaziha et al., 2014; Riedinger et al., 2011). Between them, nanoclays are the well-established components to improve the properties of hydrogel based constructs mainly due to the interactions at the clay–polymer interface (Paul & Robeson, 2008). Moreover, nanoclays can be uniformly distributed within the polymeric networks, acting as both fillers and cross-linkers agent during gel formation (Pacelli et al., 2016). Therefore, hybrid hydrogels based on nanoclays have been broadly considered in both drug delivery and tissue engineering applications (P. Li, Kim, Hui, Rhee, & Lee, 2009; Pacelli et al., 2016; Roozbahani, Kharaziha, & Emadi, 2017). The commonly used nanoclays applied for tissue engineering and drug delivery application are montmorillonite, hectorite, and laponite (belonging to the smectite family) (Wu, Gaharwar, Schexnailder, & Schmidt, 2010). The smectite family of clays is a hydrous material that swells after absorbing water and forms plastic clay with strong adhesiveness. Among the smectite family, laponite, Na0.7[(Mg5.5Li0.3)Si8O20(OH)4]0.7, is a synthetic nanoclay composed of a layered structure with 30 nm diameter and 1 nm in thickness (Wu, Gaharwar, Chan, & Schmidt, 2011). The unique properties of laponite such as high biocompatibility, anisotropic and plate-like morphology, great surface area, lack of heavy metals in its structure along with its great ability to cationic exchange make it a promising material for the improvement of numerous physical and mechanical characteristics of hydrogels in various forms (P. Li, Kim, Yoo, & Lee, 2009; H. Yang, Hua, Wang, & Wang, 2011). One of the most and interesting properties of smectite nanoclays, specifically laponite, is their blood clotting ability which  can absorb water in blood, concentrate the cells and clotting factors leading to promote hemostasis. These agents may also act as physical barriers in bleeding from damaged blood vessels and lead to coagulation (Arnaud et al., 2009; Bowman, Wang, Meledeo, Dubick, & Kheirabadi, 2011). Recent research suggested the correlation between clay surface charge and clotting efficiency due to the enhanced absorption and activation of blood coagulation factor XII on the negatively charged clay surfaces in the presence of plasma proteins (Baker, Sawvel, Zheng, & Stucky, 2007; Gaharwar et al., 2014). According to above points, it is expected that the combination of laponite nanoplatelets with IPN hydrogels of PVA-alginate may provide nanohybrid hydrogels with appropriate mechanical, physical and biological characteristics for wound healing process.
The objective of this work was to develop nanohybrid IPN hydrogels of laponite incorporated PVA-Alginate (Laponite:PVA-Alginate) and to study the effects of laponite concentration (0, 0.5, 1 and 2 wt.%) on the physical, mechanical, biological, and hemostasis properties of nanohybrid hydrogels. It is hypothesize that the nanohybrid hydrogels may have potential for tissue engineering applications specially for wound healing.
2. Materials and methods
2.1. Materials
Alginic acid sodium salt (SA) from brown algae (medium viscosity) and PVA (Mw=72,000) were purchased from Sigma-Aldrich Co. CaCl2 and methanol were obtained from Merck Co. Deionized (DI) water was used in all the sections of experiment. Synthetic silicate nanoplatelets(Laponite RDS) containing SiO2 (59.5%), MgO (27.5%), Na2O (2.8%) and Li2O (0.8%) with low heavy metals content were purchased from Rockwood Additives Limited, UK.
2.2. Fabrication of nanohybrid hydrogel of Laponite:PVA-Alginate
Nanohybrid hydrogels of Laponite:PVA-Alginate with various concentrations of laponite (0, 0.5, 1 and 2 wt.% of prepolymer) was prepared using gel casting technique. Primarily, separate alginate and PVA aqueous solutions in DI water with the concentration of 4 and 10 wt.% were prepared, respectively. After stirring at 60 °C over night, two solutions were mixed at the weight ratio of 1:1 at room temperature for 2 h to get a homogenous solution. Consequently, various amounts of laponite nanoplatelets (0, 0.5, 1, and 2 wt.%) were dispersed in 2 ml DI water and sonicated for 30 min (WUDD10H, Power 770 W), two times. Following the addition of laponite suspension to the above polymeric solution, the suspensions were mixed at 60 °C for 2 h and sonicated for 30 min, to provide a homogenous dispersion of laponite nanoplatelets. Finally, after degassing, the suspensions were transferred into the petri-dish and meaintained for 24 h to be completely prolymerized. It needs to mention that, before casting, the cylindrical glass mold was sprayed with teflon (Welcon co) for surface lubricant. Consequently, hydrogels were removed from the molds and crosslinking process was performed in two steps of covalently and ionically to crosslink PVA and alginate polymers, respectively. Initially, the hydrogels were maintained at 80 °C for 24 h, and then, dipped in absolute methanol solution for 24 h in order to crosslink PVA. Consequently, the samples were immersed in 2 wt.% CaClsolution for 24 h at room temperature to ionically crosslink alginate. After crosslinking process, uncrosslinked polymers were removed by washing the samples in phosphate buffered saline (PBS) solution. It needs to mention that, according to the concentration of laponite nanoplatelets (0, 0.5, 1, and 2 wt.%), nanohybrids were named as PVA-ALG, 0.5Laponite:PVA-Alginate, 1Laponite:PVA-Alginate and 2Laponite:PVA-Alginate, respectively.
2.3. Characterization of nanohybrid hydrogel of Laponite:PVA-Alginate
The chemical composition of the nanohybrid hydrogels was verified through X-ray diffraction (XRD, X’ Pert Pro X-ray diffractometer, Phillips, Netherlands) technique carried out with monochromatized CuKa radiation (λ = 0.154 nm) at a generator voltage of 40 kV and a current of 40 mA. Moreover, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR, Bruker tensor) performed over a range of 600-3700 cm-1 and resolution of 2 cm-1 was used to determine the chemical composition and IPN formation. Furthermore, the distance between the laponite’s layers purely and in 2Laponite:PVA-Alginate was analyzed using XRD patterns using Bragg̕ s equation (Eq. 1):
d = λ2Sinθ
In which λ is the wavelength of the copper anode source (0.154 nm), d stands for the spacing between the scattering laponite̕ s layers, and θ is the diffraction angle.
The surface morphology of the nanohybrid hydrogels as well as the distribution of laponite nanoplatelets within the IPN hydrogels was evaluated by using scanning electron microscope (SEM, Philips, XL30). Before imaging, the samples were sputter coated with a thin layer of gold. Moreover, the transmission electron microscopy (TEM, Leo 912AB) was utilized to characterize the morphology of the laponite nanoplatelets.
The swelling behavior of hybrid hydrogels were studied in PBS at 37 °C at determined time intervals. The samples were accurately weighted and immersed in PBS. After removing the samples from PBS and blotting off excess PBS, the wet mass of samples was measured. The swelling rate was calculated according to the following equation (Eq. 2):

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