Enhanced rheological behaviors of alginate hydrogels with carrageenan for extrusion-based bioprinting
Introduction
In the field of tissue engineering and regenerative medicine, several bioprinting techniques have emerged to fabricate artificial tissues and organs with precise positioning of biomaterials, biomolecules, and cells in three-dimensional (3D) space (Murphy and Atala, 2014; Das et al., 2015; Pati et al., 2016; Naghieh et al., 2018; Kim et al., 2018; Malda et al., 2013; Ozbolat and Hospodiuk, 2016). Among various biomaterials, hydrogels have been used for the most popular biomaterial to create 3D tissue-engineered structures for mimicking native extracellular matrix and providing adequate environment for cell behaviors with the appropriate porosity, pore size, interconnectivity, and biocompatibility (Malda et al., 2013; Gasperini et al., 2014; Hospodiuk et al., 2017; Radhakrishnan et al., 2016; Zhao et al., 2015). Also, the hydrogels with shear thinning properties have been most widely used in extrusion-based bioprinting because randomly arranged polymer chains of the hydrogels can be aligned and become extrudable by applying shear forces (Hospodiuk et al., 2017; Jalalvandi and Shavandi, 2019; Panwar and Tan, 2016). Therefore, it has been demonstrated in many studies that the rheological properties of the hydrogel play an important role in controlling the resolution and shape fidelity of the 3D-bioprinted structures (Kyle et al., 2017; Nakamura et al., 2010; He et al., 2016; Kang et al., 2013). Based on rheological characteristics of the hydrogels including shear thinning and thixotropic behaviors, printing parameters such as printing speed, pressure, and nozzle size can be optimized (Zhao et al., 2015; Panwar and Tan, 2016; Kang et al., 2013; Müller et al., 2015; Kesti et al., 2016; Markstedt et al., 2015).
Among several hydrogels, the alginate-based hydrogel is one of the most widely used biomaterials in the biomedical applications because of low cost and decent biocompatibility and multiple crosslinking methods (Anil et al., 2017; Venkatesan et al., 2015; Di Giuseppe et al., 2018). However, extrusion-based bioprinting of alginate suffers from low shear modulus and unstable crosslinking, which make challenging to obtain well-controlled shapes and maintain the 3D-printed structures (Radhakrishnan et al., 2016). To overcome these limitations, other additives, such as graphene oxide, silica, and cellulose nanofibers, have been used for strengthening the mechanical properties of the alginate-based hydrogels (Markstedt et al., 2015; Chung et al., 2013). Yet, these additives in the tissue-engineered scaffolds often lead to other problems related to long-term safety, biodegradability, and cost effectiveness.
Carrageenan has been widely used for gelling, thickening and stabilizing in food and pharmaceutical applications. (Vera et al., 2011; Grenha et al., 2010; van de Velde et al., 2002; Yermak and Khotimchenko, 2003; Mohamadnia et al., 2008; Popa et al., 2011). It is a natural polysaccharide extracted from red algae and consists of repeatedly (1-3)-linked β-D-galactose and (1-4)-linked α-D-galactose units (MacArtain et al., 2003). Moreover, the carrageenan is classified into three types based on the number of sulfate groups, i.e. kappa (κ), iota (ι) and lambda (λ), and these sulfate groups in the backbone provide a positive influence on cell adhesion, cell proliferation, and cell differentiation (Liu et al., 2015). Also, it was reported that carrageenan inhibits inflammatory responses because of negatively charged carboxyl and sulfate groups (Gasperini et al., 2014). In addition, the composition of carrageenan shows similarity with mammalian glycosaminoglycans in a component of extracellular matrix (Popa et al., 2015a). Due to these advantages, many researchers have used carrageenan for encapsulation of diverse cell types, drug delivery and cartilage regeneration for preclinical and clinical uses (Santo et al., 2009; Popa et al., 2012, 2015b; Rocha et al., 2011). Several studies tried to bring the advantages of carrageenan to fabricate tissue-engineered scaffolds using conventional tissue engineering techniques (Popa et al., 2011; Lim et al., 2017; Roh and Shin, 2006; Mihaila et al., 2013), but few papers regarding 3D bioprinting using carrageenan composites were published. In this study, our goal is to present the feasibility of precise fabrication of alginate/carrageenan composite scaffolds based on precise assessment of rheological properties, printability, and 3D deposition using extrusion-based 3D bioprinting. At first, the proper concentration of calcium sulfate was determined by the assessment of shear modulus of alginate-based hydrogels with various levels of ionic crosslinking. Moreover, alginate/carrageenan composite hydrogels were prepared with four different concentrations of carrageenan and used to measure their rheological properties. Based on the assessed viscosities and shear moduli of alginate and alginate/carrageenan hydrogels, printing resolutions in different printing parameters including printing speed and pressure were simulated and presented in the printability maps. In addition, alginate and alginate/carrageenan scaffolds were bioprinted with various printing parameters and used to compare their printability with the simulated results. Also, 3D deposition of both alginate and alginate/carrageenan hydrogels were assessed and compared with each other by continuous monitoring of shape fidelity in 3D structures in ten layers and similar printing resolution. Finally, the cell viability of the 3D alginate/carrageenan composite scaffolds, printed with mesenchymal stem cells using optimized printing parameters, was evaluated using live/dead staining and confocal fluorescence imaging.
Section snippets
Hydrogel preparation
Sodium alginate (Alg) was purchased from Sigma-Aldrich Inc., USA, and the final concentration of alginate solution was fixed at 2% w/v in this study. The carrageenan (Carr), mainly composed of κ-Carr, was also obtained from Sigma-Aldrich Inc., USA. The carrageenan at the concentrations of 0.5, 1.0, and 1.5% w/v was mixed with Alg solution. Calcium sulfate (CaSO4, Sigma-Aldrich Inc., USA) was used as the crosslinking agent for Alg solution. The CaSO4 solution was prepared with several
Rheological characterization of Alg-CaSO4
The frequency sweep test was carried out with different concentrations of CaSO4 with 2% alginate solution to characterize the influence of CaSO4 on the mechanical strength of the hydrogel. In Fig. 2(a), the storage modulus G′ and loss modulus G″ are shown for different concentrations of CaSO4 to determine the optimum CaSO4 concentration for highest mechanical strength. The 0% CaSO4 in alginate has lower G’ than G”, which indicates the 2% alginate is close to the liquid state at this CaSO4
Discussion
In this study, based on the simulation and measurement of rheological properties of Alg-CaSO4 and Alg-Carr-CaSO4, optimum printing parameters were explored for fabricating superior scaffolds with enhanced printability. We also demonstrated the effects of carrageenan on rheological and thixotropic properties of the alginate-based hydrogel. Therefore, the 3D-printed structure made of Alg-Carr-CaSO4 had better shape fidelity than Alg-CaSO4 after the 3D deposition of multiple layers. Eventually,
Conclusion
Our study presented a novel bioink composed of alginate and carrageenan for the extrusion-based bioprinting. The rheological properties of alginate-based hydrogel improved as the concentration of carrageenan in the composite hydrogels increased and was best when the carrageenan concentration of 1.5% was used. We optimized printing parameters based on the rheological properties and applied them on Alg-CaSO4 and Alg-Carr-CaSO4 composites. As a result, we demonstrated excellent structural strength
Acknowledgement
This work was supported by a Research Grant of Pukyong National University (2017 year).
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