Composite hydrogel gellan gum-based materials with CaCO 3 vaterite particles

. Background and Objectives : Hydrogels are cross-linked three-dimensional polymeric structures containing a large amount of water. Hydrogel materials based on natural and/or synthetic biocompatible polymers are capable of imitating the structure and properties of the extracellular matrix of living tissues. Therefore, hydrogel-based materials are widely studied and developed as functional materials in various fields of biology and medicine, including the creation of biomaterials for transplantation and tissue engineering. However, hydrogels have a number of disadvantages, such as a low biomineralization capacity, low biomechanical properties, and weak ability to form biointerface with hardtissues.Thesepropertiesmakehydrogel-basedmaterialsunsuitableforhardtissueengineering,particularly,boneregeneration.Currently, approaches to overcome these limitations, in particular, to improve the biological activity and biomineralization of hydrogels are currently being widelydeveloped. MaterialsandMethods : Thisstudyreportsanefficientapproachofhydrogelsmineralizationbasedontheultrasound-assisted synthesis of calcium carbonate CaCO 3 in the gellan gum hydrogel material. Results : The composite hydrogel materials based on the gellan gum with CaCO 3 micron-sized particles in the vaterite polymorph, uniformly distributed within the hydrogel matrix, have been obtained. The fraction of CaCO 3 in the hydrogel can easily be controlled by the number of ultrasound treatment procedures. The morphology and structure of the obtained hydrogel materials, especially the structure and distribution of the inorganic phase CaCO 3 , have been studied by scanning electron microscopy and X-ray diffraction. Conclusion : The proposed strategy for the hydrogel mineralization allows for to create functional composite materials with the potential for application for the tissue engineering, especially bone regeneration.


Introduction
Biocompatible hydrogels are among the most intensely research topics in the biomaterials development and tissue engineering due to their unique properties including structural resemblance to natural extracellular matrices [1], adjustable physicalchemical properties [2], and the fact that hydrogels can be easily incorporated by functional additives (such as inorganic materials, biomolecules and drugs) due to the high amount of water [3,4].However, hydrogels have the low ability to mineralize, and, therefore, do not support the formation of interfacial bonds with hard tissues such as bone.Mineralization of hydrogels with appropriate inorganic minerals will enhance their bioactivity, osteoconductivity and mechanical properties [5].Many examples of hydrogel mineralization have been reported so far, including mineralization with calcium phosphate [6], calcium carbonate and magnesium carbonate [7][8][9].
Among various inorganic materials, vaterite, the metastable CaCO 3 polymorph, is of particular interest due to its ability to be an efficient source of calcium ions Ca 2+ [10], rapid degradability [11], and high drug loading capacity for the various range of drugs and biologically active molecules [12,13].Moreover, vaterite promotes hydroxyapatite formation upon incubation in simulated body fluid (SBF) [14].Also it is osteoconductive and supports osteogenesis, along with widely used hydroxyapatite [15].
The gellan gum (GG) is a green naturallyderived biopolymer which is able to form waterswollen hydrogels with multivalent cations [16].Ability to absorb and retain significant volume of aqueous phase allows for efficient encapsulation of drugs.Therefore, GG hydrogels are extensively used in pharmaceutics and cosmetics as carrier and suspending agent [16], as well as biomaterial in the soft tissue engineering [17].
As a result, the composites based on the combination of biopolymer GG hydrogels with the CaCO 3 vaterite is potentially interesting materials for bone regeneration.At the moment, there are several protocols for such systems formation [9,18,19] which can be generally summarized in the following approaches: (i) mixing pre-synthesized particles into a GG solution, and (ii) particles in situ synthesis during GG hydrogel incubation in solution containing Ca 2+ and CO 2− 3 ions, including physical stimuli such as the ultrasonic (US)-assisted technique [20].This work presents the first comparative study of these various approaches to GG hydrogel mineralization and revealing of the most effective one.In the current study, the US-assisted treatment is demonstrated as a suitable and efficient technique for obtaining the composite hydrogel materials aimed at the biomedical applications.

Materials
Gellan gum (GG, Gelzan™ CM, molecular weight 200-300 kDa), CaCl 2 and Na 2 CO 3 were purchased from Sigma-Aldrich and used as received.Milli-Q water (specific resistivity higher than 18.2 MΩ cm −1 ) was obtained from a Millipore filtration system and used in all experiments.

Composite hydrogels preparation
For preparation the blank GG hydrogel, the GG aqueous solution (0.8 % wt.) was prepared as described in Ref. [21].The GG gelation was carried out by mixing of the GG and CaCl 2 (0.02 M) aqueous solutions for 30 sec at vigorous agitation and heating at +70°C.After that, the obtained mixture was cooled down to the room temperature (+22°C) and, thus, the gelation procedure was completed.
For preparation a composite GG/CaCO 3 hydrogel, the three approaches were used.
(i) Addition of preliminarily synthesized CaCO 3 vaterite microparticles to the GG solution in course of the gelation.The CaCO 3 vaterite microparticles prepared as described previously in Ref. [22] were added to the GG aqueous solution under the vigorous agitation and heating at +70°C.The agitation of obtained mixture was continued to 1 min until the homogenous distribution of CaCO 3 microparticles in the GG solution.After that, the obtained mixture was cooled down to the room temperature (+22°C).(ii) Synthesis of CaCO 3 microparticles in situ in the GG solution in course of the gelation.At the first step, an aqueous Na 2 CO 3 solution (0.33 M) was added to the GG solution under the vigorous agitation and heating at +70°C.After that, an aqueous CaCl 2 solution (0.33 M) was added to this mixture.The agitation of mixture was continued to 1 min until the completion of CaCO 3 crystallization.Then, the obtained mixture was cooled down to the room temperature (+22°C).(iii) Ultrasonic-assisted mineralization of the GG hydrogel in solutions saturated with Ca 2+ and CO 2− 3 ions.The GG hydrogel mineralization was performed using the technique of US-assisted CaCO 3 synthesis as described in the previous study [20].Hydrogel samples were immersed in an aqueous CaCl 2 solution (0.33 M) in a tube which was then placed in the ultrasonic bath.The hydrogel sample was pretreated with an ultrasound (US) (working frequency 35 kHz and radiation intensity 0.64 W/cm 2 ) in a CaCl 2 solution for 1 min.Then, an aqueous Na 2 CO 3 solution (0.33 M) was added to the CaCl 2 solution under continuous US treatment.The synthesis of CaCO 3 particles in the GG hydrogel was carried out for 1 min in the presence of US treatment.
All the obtained GG hydrogel materials were stored in a refrigerator at the temperature of +5°C.

Characterization methods
The surface morphology of hydrogel materials was studied by the scanning electron microscopy (SEM) with using a Tescan MIRA II LMU setup (Tescan, Czech Republic).The hydrogel mass was monitored with a Sartorius Quintix 35-18 (Germany) microbalance prior to US-assisted mineralization treatment, after each mineralization procedure, and after following drying in a drying oven for 4 hours, at +60°C in air.Powder X-ray diffraction analysis of the preliminary dried and grinded samples was performed with a Rigaku Minilex-600 diffractometer (Rigaku Corporation, Tokyo, Japan) using Cu-K α radiation (40 kV, 15 mA, Ni-K β filter) in the 2θ range 5-60°at a scan speed 1°/min.The XRD data obtained were compared with the literature crystallographic data for vaterite [23] and calcite [24].
The average diameter of CaCO 3 particles and the filling factor of CaCO 3 in composite GG hydrogels were measured by analyzing SEM images with the use of the Image J software (https://imagej.nih.gov/ij/index.html).The areas occupied with CaCO 3 were extracted from the total area of the hydrogel cross-section by the area selection function.Then, the filling factor was calculated as the ratio of the CaCO 3 -filled area to the total area [25].

Results and discussion
Hydrogel matrices based on anionic polysaccharide gellan gum were prepared by GG ionotropic gelation with the divalent calcium ions Ca 2+ as a cross-linking agent, which is promote the site binding of carboxylate groups of GG polysaccharide helical chains [26].Moreover, the addition of calcium ions lowers the negative charge of the GG helical chains and, thus, facilitate their electrostatic internal interaction and leading to gelation as well [26,27].In this way, a three-dimensional network of a water-insoluble GG polysaccharide hydrogel is formed.Photo and SEM images of the resulting GG hydrogel are shown in Fig. 1, a, b and  c, respectively.
The modification of GG hydrogels by CaCO 3 microparticles was performed using three different approaches.A comparison study of these approaches allows to optimize the method of hydrogels mineralization, and, thus, to obtain composite hydrogel materials with the most optimal structural and functional characteristics, particularly, with CaCO 3 in the vaterite polymorph.Vaterite is able to provide the required functionality of a composite material, including Ca 2+ release capacity [10] and high drug loading efficiency [13], which is significantly important for biomedical applications, especially for bone tissue engineering [28].
In the first approach, CaCO 3 microparticles were added to GG aqueous solution under rapid homogenization of the mixture (Fig. 1, d).In this case, the presence of water environment initiates the process of partial dissolution of CaCO 3 .Therefore, an amount of free Ca 2+ ions are occurred in the solution near the surface of the CaCO 3 particles, which promotes the ionotropic gelation of GG.Thus, the addition of CaCl 2 for gelation is not required in this case.It can be observed that the CaCO 3 microparticles in the hydrogel have the form of cubic crystals, which indicates that the vaterite has recrystallized into calcite [29] (Fig. 1, e, f ) because of the dissolution process.The CaCO 3 particles are distributed in the hydrogel in the form of agglomerates.
In the second approach, a Na 2 CO 3 aqueous solution was added to GG solution for providing carbonate CO 2− 3 ions which are necessary for the growth of CaCO 3 microparticles.Further, the following addition of Ca 2+ ions to the mixture initiates two parallel processes.First, the electrostatic and site binding of GG molecular chains by calcium ions is resulting in the gelation of GG.Second, the interaction with carbonate ions is resulting in the formation of CaCO 3 particles in GG hydrogel.In this way, the "embedding" of CaCO 3 inorganic particles into the GG hydrogel matrix occurs during its gelation and results in the formation of a composite material.One can observe the inhomogeneous distribution of the CaCO 3 inorganic phase in the hydrogel (Fig. 1, g).The CaCO 3 exhibits cubic crystals characteristic for calcite polymorph [29] as well as an amorphous [30] phase (Fig. 1, h, i).
In the third approach, the pre-formed GG hydrogel was pretreated in a CaCl 2 solution under ultrasound irradiation, which initiates the diffusion of Ca 2+ ions from solution into the hydrogel network.The following addition of carbonate CO 2− 3 ions initiates the US-assisted growth of CaCO 3 in the GG hydrogel.The scheme of US-assisted process of hydrogel modification is shown in the Fig. 2. The spherical particles with a porous surface morphology, which is typical for vaterite polymorph, as well as a uniform distribution of particles in the hydrogel can be observed (Fig. 1, j, k, l).
Thus, a comparison of the structure of composite GG hydrogel materials formed by different methods allows us to conclude that the US-assisted mineralization provides the most optimal structure of a composite material with respect to the polymorphic modification of CaCO 3 microparticles and their distribution in the material.In terms of functionality, CaCO 3 in the polymorphic modification of vaterite is preferable to calcite, since it has a higher bioactivity, osteogenic potential [28], and a higher capacity for immobilization of biologically active moieties [13].
Further, to study the effect of the US-assisted CaCO 3 synthesis on the composite material structure, three procedures of the such US treatment of GG hydrogels were carried out.On SEM images of hydrogel samples after 1 st , 2 nd , and 3 rd US treatment procedures (Fig. 3, a), it can be observed that as a result of an increase in the number of procedures, the size and amount of CaCO 3 particles in the material increase as well (Fig. 4, a, b).The statistically significant increase in average diameter of CaCO 3 particles occurs after 3 rd US treatment procedure, while filling factor drastically increases already after 2 nd procedure.After the third procedure, the cubic calcite microparticles are observed in the hydrogel.The weight of samples (both wet and dried) showed that the water content in the hydrogels increased (Fig. 4, c) along the statistically significant decrease of dry mass (Fig. 4, d) after the 1 st US treatment procedure.It is worth noting that there is no statistically significant difference in changing of dry mass amount for samples after 1 st , 2 nd and 3 rd US treatment procedures.Also, the following  (2 nd and 3 rd ) treatment procedures did not cause any statistically significant change in the water content in the hydrogels.The diffraction patterns of these samples (Fig. 3, c) show peaks characteristic of vaterite, which confirm the presence of CaCO 3 in the vaterite polymorph in the composite material.The diffraction patterns for samples after second and third procedures show the presence of calcite peaks as well.Thus, one can conclude that a consistent increase in the number of US treatment procedures of GG hydrogel initiate the process of recrystallization of vaterite particles into calcite.Therefore, the two US treatment procedures were considered as opti-mal for the formation of a stable CaCO 3 phase in composite GG hydrogel, from the point of view of the vaterite polymorph of CaCO 3 and particles distribution in the hydrogel matrix.

Conclusions
In the presented study, gellan gum hydrogels have been functionalized with CaCO 3 microparticles in the porous vaterite polymorphic form.The comparative study of three approaches to the formation of a composite material based on gellan gum hydrogel and CaCO 3 microparticles has been performed.The structure, polymorphic modification of CaCO 3 particles, as well as their distribution in the hydrogel matrix depend on the conditions of their synthesis.It has been shown that the most efficient approach of hydrogel mineralization is the ultrasonic (US)-assisted treatment of GG hydrogel in Ca 2+ and CO 2− 3 containing solutions.The US treatment stimulates penetration of these ions into the polymeric hydrogel network and facilitates porous vaterite microparticles formation inside the hydrogel.It has been found that the presence of ultrasonic treatment during the CaCO 3 particles synthesis allows to obtain particles with the necessary polymorph (vaterite) and a uniform distribution in the GG hydrogel.The effect of the US treatment procedures number on the structure of the composite GG hydrogel has also been studied, and it has been shown that the two procedures are optimal in terms of the structure and distribution of CaCO 3 particles in the hydrogel matrix.Composite GG hydrogels functionalized with vaterite can be promising for application as the implantable materials for bone tissue regeneration.

Fig. 1 .
Fig.1.a -photo of the blank GG hydrogel, b and c -SEM images of the blank GG hydrogel; d -photo of the composite GG hydrogel in a petri dish, prepared by the addition of preliminarily synthesized CaCO 3 microparticles to the GG solution, e and f -SEM images of this composite GG hydrogel; g -photo of the composite GG hydrogel in a petri dish, prepared by the in situ synthesis of CaCO 3 microparticles in the GG solution in course of the gelification, h and i -SEM images of this composite GG hydrogel; j -photo of the composite GG hydrogel, prepared by the ultrasonic-assisted mineralization of the GG hydrogel in solution saturated with Ca 2+ and CO 2− 3 ions, k and l -SEM images of this composite GG hydrogel (color online)

Fig. 2 .
Fig. 2. Scheme of the process of ultrasonic mineralization of GG hydrogel by CaCO 3 vaterite particles (color online)

Fig. 3 .
Fig. 3. a -photo and corresponding SEM images of initial GG hydrogels, and composite GG hydrogels after one, two and three US-assisted CaCO 3 mineralization procedures, b -diffraction patterns of composite GG hydrogels after one, two and three US-assisted CaCO 3 mineralization procedures (color online)