Next Article in Journal
Alkenyl Succinic Anhydride: The Question of Covalent Bonding and Chemistry Considerations for Better Sizing—Review
Next Article in Special Issue
Advanced Bioresin Formulation for 3D-Printed Bone Scaffolds: PCLDMA and p-PLA Integration
Previous Article in Journal
Designing Sustainable Polymer Blends: Tailoring Mechanical Properties and Degradation Behaviour in PHB/PLA/PCL Blends in a Seawater Environment
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Polymers
Volume 15
Issue 13
10.3390/polym15132875
polymers-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Experimental Advances in the Real-Time Recording of Cross-Linking Alginate In Situ Gelation: A Review

by
Ioanna N. Besiri
,
Thomas B. Goudoulas
*,
Ehsan Fattahi
and
Thomas Becker
Research Group of Fluid Dynamics, Chair of Brewing and Beverage Technology, TUM School of Life Sciences, Technical University of Munich, Weihenstephaner Steig 20, 85354 Freising, Germany
*
Author to whom correspondence should be addressed.
Polymers 2023, 15(13), 2875; https://doi.org/10.3390/polym15132875
Submission received: 6 June 2023 / Revised: 26 June 2023 / Accepted: 27 June 2023 / Published: 29 June 2023
(This article belongs to the Special Issue Advances in Rheology of Bio-Based Polymeric Materials)
Browse Figures
Versions Notes

Abstract

:
Alginate-based hydrogels are promising smart materials widely employed in the food, bioengineering, and energy sectors. The development and optimization of their production require a thorough knowledge of gelation. In recent years, advanced experimental procedures have been developed for real-time cross-linking alginate reaction monitoring. Novel methods, such as customized rheometric setups, enable the recording of mechanical properties and morphological changes during hydrogel formation. These innovative techniques provide important insights into the gelation stages, the reaction rate, the diffusion of cross-linker to polymer chains, and the homogeneity of the gelling structures. Based on real-time experimental data, kinetic models are developed to enhance comprehension of the reaction mechanism and, eventually, to predict the gelation progress. The aim is to enable better control of the characterization of both the complex gelation and the propagated structures. This review aspires to present a comprehensive overview and evaluation of the breakthrough innovations of the real-time in situ recording of cross-linking alginate hydrogels and bead formation. A detailed analysis of the pioneering experimental developments provides a deep comprehension of the alginate gelation, including the parameters controlling the reaction.

1. Introduction

The favorable characteristics of alginate, such as biocompatibility, biodegradability, and antimicrobial properties [1,2,3], render it an ideal material for developing ‘smart gelling’ products with a wide range of applications in bioengineering, food, and energy domains [4,5,6]. The polysaccharide is primarily extracted from the cell walls of brown seaweeds [7,8]. To easily tailor the structure and characteristics of the macromolecule, biological production by two genera of soil bacteria, i.e., Azotobacter and Pseudomonas, is feasible as well [9,10]. Alginate is composed of (1–4)-linked β-D-mannuronic (M) and α-L-guluronic (G) acid monomers arranged in different sequences, i.e., MM-, GG-, and MG-, along the negatively charged long linear chain, as shown in Figure 1a [11,12]. Gelling networks are formed in the presence of alginate in aqueous solutions [13,14]. At a pH lower than the pKa of uronic acid, hydrogels are formed, while the reaction of the polysaccharide with divalent cations results in well-known ionic hydrogels [15,16]. The ionotropic gelation is based on the ‘egg-box′ model, presented in Figure 1b,c, which arises from the initial chelation of cations with the alginate chains and the following interchain association, [17]. It critically depends on the physicochemical characteristics of the reactants, such as their concentration, the kind of cations, and the molecular weight, Mw, and M/G of alginate, as shown in Figure 2 [18]. The divalent metals bind mainly with G- and MG- sequences of the polymer backbone through physical cross-linking [19,20,21]. The dimerization of alginate chains is induced, and multimer structures are formed following the zipper mechanism [22,23]. The selectivity of the polysaccharide with cations increases in the order Mg2+ < Co2+, Ni2+, Zn2+ < Ca2+ < Sr2+ < Ba2+ < Cd2+ < Cu2+ < Pb2+ [24,25]. The reaction is controlled by the stoichiometric mole ratio f = [X2+]/[COO−], where X2+ represents the divalent cations. As f increases, the gelation is faster, and stiffer structures are formed [26,27]. Moreover, the alginate gelation can originate from chemical cross-linking. For instance, some bivalent cations and trivalent metals, as well as other chemical intermediates such as epichlorohydrin, cross-link with alginate chains through strong covalent bonds [20,21]. Additionally, to fabricate materials with enhanced mechanical strength, alginate can create double network (DN) hydrogels with other macromolecules, such as gelatin or polyacrylamide [28,29].
The breakthrough applications of hydrogels dictate an extensive comprehension of the formation mechanism [30,31]. Advanced methods, such as rheology and light-triggered techniques, allow for recording of the gelation kinetics in situ [32,33]. Monitoring the process determines the stages of the reaction, the factors which evolve in each stage, and the temporal micro-structural rearrangements [34,35]. Based on real-time experimental data, kinetic models are developed to enable a deeper understanding and accurate prediction of the gelation progress. This leads to the development of strategies to optimize the characterization of complex gelation [36,37]. The present review demonstrates and evaluates the advanced in situ methods for monitoring the alginate cross-linking gelation. Novel concepts based on rheology and time-resolved techniques, such as small angle X-ray scattering (SAXS), monitor the alginate hydrogel and bead composition. Information is provided on the temporal evolution of the mechanical properties of the system to detect the dominant parameters affecting the reaction and further elaborate the diffusion mechanism of the cross-linker to alginate solution. Additionally, the visualization of gelation captures the changes in the gelling structures and investigates their homogeneity. With a thorough assessment of the in situ methods, the challenges and opportunities of real-time gelation recording are discussed to shed light on potential future research that could further advance this domain.
Polymers 15 02875 g001
Figure 1. (a) Schematic representation of mannuronic (M) and guluronic (G) acid monomers. (b) Graphical illustration of alginic acid consisting of MM-, GG-, and MG- sequences. (c) The reaction of cations (X2+) with alginate chains results in the formation of ‘egg-box′ structures [38]. Reproduced with permission from ACS, Urbanova et al. (2019), Biomacromolecules, 20, 11, 4158–4170 (https://pubs.acs.org/doi/10.1021/acs.biomac.9b01052). Further permissions inquiries related to the material excerpted should be directed to the ACS.
Figure 1. (a) Schematic representation of mannuronic (M) and guluronic (G) acid monomers. (b) Graphical illustration of alginic acid consisting of MM-, GG-, and MG- sequences. (c) The reaction of cations (X2+) with alginate chains results in the formation of ‘egg-box′ structures [38]. Reproduced with permission from ACS, Urbanova et al. (2019), Biomacromolecules, 20, 11, 4158–4170 (https://pubs.acs.org/doi/10.1021/acs.biomac.9b01052). Further permissions inquiries related to the material excerpted should be directed to the ACS.
Polymers 15 02875 g001
Polymers 15 02875 g002
Figure 2. Parameters determining the ionotropic alginate gelation.
Figure 2. Parameters determining the ionotropic alginate gelation.
Polymers 15 02875 g002

2. Introduction to In Situ Gelation Methods

The innovative applications of polymers require detailed knowledge of their molecular characteristics and reaction mechanisms [39,40]. The conventional ex situ preparation of hydrogels limits their study to a steady-state of kinetics, impeding a comprehensive investigation of their dynamic structural and mechanical properties [41]. In contrast, the in situ methods provide an opportunity to record the gelation in its original position from the initiation of the reaction [42,43]. As Figure 3 shows, in situ gelation can facilitate the precise control of the volume and concentration of reactants to modulate the reaction accurately. Furthermore, the detection of gel point, i.e., the instant that the transition from solution to gel phase occurs [44], is achieved along with the recording of the dynamic mechanical response and structural development of hydrogels. In this way, the kinetic stages and the parameters defining each reaction step are determined. Finally, kinetic models can be developed based on real-time experimental data to describe and predict the reaction.
Table 1, which is at the end of the review, includes the methods developed during the last years to characterize the cross-linking alginate in situ gelation. In brief, the mechanical properties of alginates are controlled by advanced rheometric and light-triggered techniques, as well as by cantilever sensors. Moreover, innovative procedures based on time-resolved methods, such as small-angle Χ-ray scattering, Fourier-transform infrared spectroscopy (FTIR), and dark field microscopy (DFM), are designed to detect the microstructural and macrostructural rearrangements during the gelation. A detailed description of these techniques is presented in Section 3 and Section 4.
In situ experimental procedures are used to form alginate hydrogels or alginate beads. The hydrogels are generated by the diffusion of the cross-linking agent into alginate solution [45], while the beads derive from the drop-wise methods in which alginate solution drops into pools of gelling solution [46]. The source of the cross-linker determines whether the gelation is internal or external [47]. In the first case, insoluble salt is added to the alginate solution, and the cations diffuse to the chains of the polymer when the appropriate pH conditions are achieved by a catalyst such as D-glucono-δ-lactone (GDL). The slow hydrolysis of GDL releases the protons, and they react with the biopolymer [48,49]. In the second case, the gelation is achieved by the direct exposure of alginate solution to an active form of cations, specifically, a soluble salt [50,51]. The external gelation implies a faster reaction than the internal mechanism, but the slower internal gelation leads to more homogeneous gelling structures [52,53].

3. In Situ Real-Time Recording of Alginate Hydrogel Formation

3.1. Conventional Rheometric Setups

Real-time recording of gelation kinetics provides valuable insights into the cross-linking reactions of alginate during hydrogel formation. Rheology, the study investigating the flow of matter [54], is extensively used to evaluate the progress of gelation in dynamically developing systems [55,56]. Specifically, oscillatory time sweeps are performed to study the temporal evolution of the mechanical properties of materials undergoing time-dependent structural rearrangements [57]. The mechanical properties are represented by the storage (G′) and loss (G″) moduli, showing the elastic and the viscous responses of the system, respectively. When G″ > G′, the material behaves as a solution, while when G″ < G′, the gel behavior predominates [54,55,57]. At the crossover point of the moduli, the first microgel structures are created and the reaction initiates [54]. For this reason, it can be called the gel point, which is accurately determined by in situ rheological experiments, leading to the detection of the precise gelation time, in contrast to the vial inversion test, which detects gelation time by inverting the vials until the material no longer flows [58,59]. Over the years, the most common method for conducting these dynamic experiments has been the utilization of conventional rheometric plates [34,35,60]. Typically, a uniform mixture of reactants is prepared ex situ, and it is then applied to the bottom plate of the rheometer directly after mixing, where it is subjected to dynamic oscillatory tests [61,62]. The recording of modulus kinetics is effective and successful [63,64]. This rheological procedure is mainly applied to study internal gelation, as the slow reaction provides enough time for sample placement and initiation of the oscillatory experiments within the solution state of the material [65].
The study of Funami et al. [66] is remarkable, utilizing internal gelation to investigate the kinetics under different physicochemical characteristics of reactants. Alginate hydrogels rich in G-residues are more elastic in the presence of higher CaCO3 concentrations (15 mM), while the elasticity increases in those rich in M-residues in the lowest availability of cations (3.75 mM). They obtain rod-like and network-like conformations, respectively, as shown in the additional atomic force microscopy images. Based on long-term oscillatory time sweeps, the authors propose the following first-order kinetic equation to describe the temporal evolution of the storage modulus, G′:
G ′ ( t ) = G s a t ′ { 1 − exp [ − k t ( t − t 0 ) ] } ,
Equation (1) has been applied to determine the gelation time (t0), the rate constant (kt), and the saturated storage modulus ( G sat ′ ) of alginate hydrogels, i.e., the equilibrium modulus as t reaches infinity [66,67]. In their following research work, a simpler equation with one exponential factor was employed to describe the internal alginate gelation adequately [67].
Similarly, Fernández Farrés et al. [68] have investigated the in situ formation of alginate-Ca2+ fluid hydrogels using a four bladed vane in cup on a Kinexus Rheometer to control the shear profile. During the reaction, the viscosity increased due to the propagation of ‘egg-box′ structures, while the shear rate caused a break-up in the network, and micro-gelled particles were formed. Under constant concentrations of reactants, the lower shearing (100 and 200 s−1) results in higher viscosities, while the higher ones (400 and 600 s−1) may cause a delay in the association of the initially formed particles and decrease the reaction rate. Based on real-time data of G′ obtained by dynamic oscillatory experiments with cone-and-plate geometry directly after the formation of structures at different shear rates, kinetic models are proposed to describe the gelation. An exponential equation applied to the high-Mw alginate hydrogels shows that the kinetic constant is the same for all structures, meaning that, at the time of shear removal, the strength rate is independent of the external forces, since the processing period is higher than the gelation time. For the reaction of low-Mw alginates, a linear first-order model has been employed, revealing that the ordering rate increases when hydrogels are formed at low shear rates, which accelerates the development of particles [68].
Another interesting study reports a customized mixing of reactants by developing a syringe-induced technique, one which utilizes internal gelation [69]. In brief, Larsen et al. [69] prepare one syringe containing soluble alginate and one with insoluble alginate/gelling ion particles, as shown in Figure 4a,b. Through a three-way connector, as presented in Figure 4c, the soluble alginate is transferred to the other syringe, and, upon mixing of reactants, it is placed through a channel onto the standard plate of the rheometer to record the gelation which occurs according to the mechanism in Figure 4d,e. The effect of cations on gelation kinetics can be controlled, and the proposed exponential kinetic equation, describing the temporal evolution of G′, shows that the gelling rate is determined by the release rate of cations from the particles [69].
Furthermore, the conventional rheometric setups serve to study the temperature control kinetics of cross-linking alginate gelation due to the attached Peltier system [70,71]. Goudoulas et al. [72] have successfully studied the phase-transition kinetics of binary alginate–gelatin mixtures by applying isothermal time sweeps in a temperature range (5–20 °C) using a parallel-plate configuration on an Anton Paar MCR 502 rheometer. Additionally, the standard rheometric configurations can record the reaction of alginate with a covalent cross-linking agent upon ex situ mixing of reactants, since the sol–gel transition occurs relatively slowly. This has been achieved by Li et al. [73] in order to study the reaction of N,O-carboxymethyl chitosan with oxidized alginate without additional cross-linkers, as well as by Yang et al. [74] to record the gelation of alginate with adipic acid dihydrazide through coupling agents. However, they cannot provide adequate monitoring of the external gelation, which occurs instantaneously, i.e., within the first seconds of mixing the reactants.

3.2. Advanced Rheometric Setups

Advanced rheometric methods have been designed to enhance the investigation of the mechanical response in diffusion-controlled alginate hydrogels [75]; for instance, when crafted polysaccharide is exposed to light, conformational changes occur, resulting in gelling networks [76]; Bonino et al. [77] have utilized the light emitting diode (LED) bottom plate of a TA stress-controlled rheometer to monitor the gelation of alginate chemically modified with methacrylate groups when a photoinitiator presents in the system. This method facilitates the detection of the gel point and the evaluation of the reaction kinetics. Higher UV intensities accelerate the sol–gel transition and increase the storage (G′) and loss (G″) moduli at a constant concentration of the system. Additionally, based on short-term UV oscillatory experiments, an empirical exponential model is applied tο obtain the moduli at steady-state, which is found to be critically dependent on the degree of methacrylation [77]. Subsequently, the same research group observed that in addition to cross-linking due to the light-induced method, there is evidence of micro-structural development in the absence of UV light, i.e., dark curing. This is attributed to the acidic pH of the mixture, which facilitates the CaCO3 dissociation and the cross-linking with alginate chains. Subjected or not to dark curing, the final properties of gelling formations are unaffected, reaching the same modulus plateau, but the gelation rate slows down when UV is not continuously applied to the gelation [78].
The external cationic alginate gelation exhibits rapid kinetics and forms heterogeneous structures, facts making it challenging to in situ monitor the mechanical properties using the conventional rheometric plates. To address this issue, innovative customized rheometric setups have been developed. In 2016, Mahdi et al. [79] demonstrated a modified lower rheometric plate, as presented in Figure 5a, facilitating the initiation of reaction at the instrument. It consists of a petri dish with filter paper impregnated with CaCl2 solution and a hydrated dialysis membrane on the top. The alginate solution is placed there, preventing its absorption by the filter paper. The fast initiation and the subsequent slower evolvement of the reaction can effectively be recorded, as shown in Figure 5b. Additionally, the systematic increase of moduli in the presence of more cations in the system can be recorded. Interestingly, the design enables the monitoring of hydrogel degradation by replacing the CaCl2 filter with one containing a calcium chelator [79], while it has been utilized to study the effect of the physiological fluids on the mechanical response of cross-linking hydrogels [80]. Based on these advantages, a rheo-dissolution cell has been developed controlling both the in situ gelation and the drug release profile of gelling systems [81].
In 2020, Besiri et al. [82] introduced an advanced setup for mixing the reactants on the rheometer during the external gelation, one resembling a batch reactor operation. It consists of two parts with an inner void when coupled together. A cross-linking agent fills the internal space with a volumetric syringe connected to a side-feed hole. At the initiation of the rheological measurement, CaCl2 is injected through micro-holes into the alginate solution placed on the top of the setup, and the reaction begins. As shown in Figure 6a,c, the design can be easily adaptable to various rheometers with a flexible arrangement of micro-holes to study the process thoroughly. Figure 6b presents that the setup can successfully record the initial and quasi-steady-state kinetic stages and assists in the determination of the fast sol–gel transition, i.e., the crossover of moduli, where the first gelling structures are created. Additionally, the stoichiometry of reactants can be monitored. Higher concentrations and injected volumes of CaCl2 increase the growth rate of G′, signifying that the diffusion of cations controls the gelation. By controlling the angular frequency (ω) it is shown in Figure 6d that higher ω results in a sharper increase of G′, i.e., the formation of stiffer hydrogels, which is attributed to the acceleration of the propagation of multimer structures. The configuration can form typical soft hydrogels after 1 h of reaction, as it is observed in Figure 6e by frequency sweep experiments where the moduli are developed at a slow rate independently of the applied ω [82,83]. Further, in situ investigation demonstrates that alginates with higher Mw and M/G increase the elasticity of the network due to the higher availability of free polymer chains for cross-linking, as shown in Figure 7 [84]. Currently, the novel setup is utilized to provide insights into external alginate gelation concerning the spatial distribution of the ions’, while a two-kernel equation, consisting of one exponential and one logarithmic factor, seems to describe and predict the reaction adequately [85].
In addition to the innovative rheometric designs, interfacial shear rheology has been utilized to control the in situ generation of alginate foam hydrogels [86]. Ben Djemaa et al. [86] propose a gas-induced cross-linking reaction instead of the conventional internal mechanism for hydrogel formation. Gas steam, supplied with CO2 through a flow meter, is humidified and connected to a double-wall ring geometry containing an alginate solution with CaCO3 particles. The flow of CO2 in the interface of the pre-gelling system triggers the gelation. G′ and G″ increase rapidly within 90 min, since the diffusion of CO2 causes the acidification of the interface, providing the suitable pH conditions for the cations’ release to alginate chains. Later, the moduli tend to reach a plateau, possibly meaning that the acidification process and the gel front have been developed in the entire system, i.e., from the interface to the bottom of the trough. The researchers state that the physicochemical characteristics of reactants and the CO2/air ratio determine the gelation progress and need further investigation [86].

3.3. Alternative Techniques beyond Rheology

Apart from employing customized rheometric techniques to understand alginate gelation, researchers propose to use cantilever sensors, as well. A cantilever sensor consists of a beam anchored at one end and having a free extension into space, and it is made from a thin film with mechanical flexibility. The movement of the beam, along with the combination of piezoelectric elements, enables the detection of the deflection of the cantilever, which results in the measurement of the mechanical response of materials. This experimental approach minimizes the time required for monitoring the dynamic properties, while the gelation can occur under high frequencies [87]. Recently, Haring et al. [88] have evaluated alginate-Ca2+ kinetics with a piezoelectric-excited millimeter cantilever (PEMC) sensor [89]. In the implementation, it is immersed in a petri dish containing alginate, in which CaCl2 is injected 5 mm from the anchor of the cantilever upon stabilization of the signal. In this way, the resonant frequency is recorded and used to calculate the G′ and G″ of the developed gelling structures through fluid–structure interaction models. The gelation kinetics can be evaluated similarly to oscillatory rheological experiments, but the crossover of moduli is not detected due to the high frequency, i.e., G′ > G″ from the reaction’s initiation [88].
The overall comprehension of the cross-linking gelation requires not only the recording of mechanical properties but also the investigation of the structural conformation [90,91]. Yamamoto et al. [92] have designed a customized sampling system combined with SAXS to in situ monitor the polymer chains’ association in alginate–Ca2+ structures developed through competitive ligand exchange (CLEX) cross-linking [93,94]. After mixing two alginate solutions, the one containing a cross-linking agent (CaEDTA) and the other an exchange ion (ZnEDDA), Zn2+ ions are exchanged with Ca2+ ions, which are subsequently available to cross-link with polymer chains. As shown in Figure 8a, syringe pumps injected the solutions in a sample cell so that an interface between them was created, and the reaction was controlled only by the diffusion of reactants. The X-ray irradiated the interface in short time intervals to continuously monitor the process. The Kratky plots presented in Figure 8b show that q2I increases linearly with q (I the intensity, and q the magnitude of scattering), meaning that the polymer chain is in a single coil condition at pH = 7, while the maximum value at 0.5 nm−1 indicates the chain association in the first 2 min of the experiment. The researchers have observed that, at higher pH, the signal of SAXS is weaker due to higher aggregation and less cross-links of alginate chains. Based on this experimental data, a two-component broken rod model is proposed to evaluate the cross-sectional form of junction zones [92]. Recently, Pragya et al. [95] probed the dynamic structures of an alginate–acrylamide double network in the presence of both cationic and covalent cross-linking agents by applying a series of time-induced methods, such as FTIR, UV-vis and Raman spectroscopy. In situ hydrogel characterization after the direct mixing of reactants indicates that the gelation is developed in two phases. Initially, physical entanglements of polymers and covalent cross-linking occur. Later, the cross-linking interactions control the reaction [95].
In situ visualization of the process provides important insights into the morphological rearrangements during the reaction [96,97]. Chueh et al. [98] have observed the formation of hydrogels in microchannels upon the release of cations to alginate chains from a caged calcium complex through UV light exposure. The non-gelled alginate is removed from the system through washing, and the gelling structures are dissolved in the presence of EDTA, resulting in the recovery of the flow in channels. The method controls the flow of the system in microfluidic devices applicable to in vitro 3D cell culture. Similarly, Braschler et al. [99,100] have monitored the gel front in an alginate–Ca2+ system with a sequential gel layer synthesis in a microfluidic chip, using fluorescent polymer combined with the theoretical study. They reported that the diffusive depletion zone affects the concentration profile on the gel front, while higher flow rates decrease the gel’s growth rate. This may be attributed to an interplay between the more alginate chains diffusing in the gel front, which require more Ca2+ ions, and the washing out of partially cross-linked chains during the deposition of layers. Overall, these decrease the concentration of cations in the reaction zone [99,100]. Nunamaker et al. [101] have visualized diffusion-based gelation by dropping CaCl2 into an alginate solution consisting of a complex indicator. The gel rate is detected by the color change of the indicator from dark red to yellow in 50% of hydrogel, and it is influenced by the physicochemical properties of reactants, i.e., the higher the cationic concentration, the faster the gel’s development.
Interestingly, Secchi et al. [102] have investigated the microscopic dynamics of cross-linking gelation by applying photo correlation imaging (PCI), a light scattering method, on a gelling system in which CaCl2 slowly permeates through a membrane to an alginate solution. Additionally, the speckle motion in PCI controls the local flow field within the sample, revealing that the gel front is regulated by the diffusion of cations and molecules and critically depends on the convection of reactants through the local flow [102]. Another captivating research effort utilizes dark field microscopy (DFM) to record the progress of the gel front during an alginate cross-linking reaction [103,104]. As shown in Figure 9a, two microscope slides with an alginate droplet in the center, in the gap between them, are gently pressed together to create a flow cell. CaCl2 is injected in this space at the periphery of the cell, and the diffusion-controlled reaction initiates. The flux of cations occurs in the radial direction, as Figure 9b shows. In this way, the gel front increases from the periphery to the center of alginate droplet during the cross-linking reaction, as can be observed in the DFM images in Figure 9d. The difference between gelled, i.e., narrow bright band, and ungelled alginate is distinct. This visualization is utilized to calculate the velocity of the gel front. Figure 9c shows that the gel front velocity is higher when more cations are available. In this way, the distance of non-gelled alginate solution from the periphery of the cell sharply increases due to the high number of cross-links formed in the reaction zone. Finally, a reaction–diffusion model is applied to describe and predict the dynamic gel front for a variety of geometries in which the gelation is developed [104].

3.4. In Situ Alginate Hydrogels in Membranes

The in situ control of cross-linking alginate hydrogels in membranes provides important information for their filtration, which is advantageous for industrial and environmental applications [105]. Hassan et al. [106] have obtained the pseudo-first-order kinetics of multimembrane alginate hydrogels in capillary columns at different temperatures. The gelation is developed rapidly in the beginning and at a slow rate across longer times due to the high density of the network of the gelled membrane [106]. Additionally, Sioutopoulos et al. [107] have investigated in situ the mechanical properties of fouled alginate segments on reverse osmosis membranes using a stress-controlled rheometer. The researchers reported that the elasticity increases when low concentration of cations flow to the membrane, possibly due to additional strong cross-links of Na+ with alginate macromolecule [107]. Epstein et al. [108] have recently proposed an advanced configuration combining micro-rheology with microcopy to control both the mechanical and conformational changes of a foulant layer at a customized alginate–Ca2+ membrane cell connected to a pressurized feed vessel. The mechanical properties are calculated by the particle-tracking method. At higher fluxes, compaction of the foulant layer occurs, and stiffening of the material is observed [108].

4. In Situ Real-Time Recording of Alginate Bead Formation

The broad implementation of alginate beads in environmental and biological applications highlights their significance [109,110]. Electrodispersion reactors and electrospray methods are only a few of the techniques applied to form cross-linking beads [111,112]. The enhanced comprehension of their synthesis makes the in situ control of their reaction kinetics imperative in order to evaluate their dynamic properties [113,114,115]. Lee et al. [116] injected an alginate droplet into baths of calcium sources with mixing times up to 4000 s. An osmotic gradient is created between the beads and the solution bath, which is responsible for cation diffusion to the gelling matrix. By rheological investigation of beads of 8 mm diameter, it was observed that, after 100 s of mixing, the mechanical properties of alginate–CaCl2 beads were constant, meaning that the preparation time is short compared to this of calcium lactate and calcium gluconate sources [116]. Additionally, a microfluidic flow-focusing device was employed to produce alginate microparticles, which were then injected into a CaCl2 tank. When continuous stirring was applied, the diffusion of cations to alginate particles was accelerated, and their faster solidification was achieved, as opposed to the particles gelled in the non-stirred tank [117].
Moreover, visualized extrusion-dripping methods are utilized to capture the shape and size of alginate–Ca2+ beads, which are affected by the physicochemical characteristics of solutions, the size of the droplets, and the collecting distance [118,119]. Haldar et al. [120,121,122] have developed the configuration presented in Figure 10a. As alginate drops into a calcium solution bath, a high-speed imaging system records the formation of beads. As observed in the sequential snapshots of Figure 10b during gelation, the crater is propagated in the liquid surface of cross-linking agent, while the dynamic structural arrangements are distinct. The crater is restored to its shape upon the injection of the alginate droplet into the CaCl2 solution [120].
Furthermore, Stößlein et al. [123] have constructed a customized sample holder on a texture analyzer, as depicted in Figure 11a. The configuration creates inhomogeneous alginate beads with an alginate–Ca2+ shell and a core of pure cross-linking agent. Upon the beads’ formation, the beads are pressurized, and their mechanical response is obtained as a function of time, as shown in Figure 11b. At approx. 300 s, a plateau was recorded indicating the two stages of kinetics. The authors also observed a decrease in the volume at 300 s, indicating that the fast gelation will not occur after 300 s [123].
Recently, Posbeyikian et al. [124] have controlled the gel front in alginate–Ca2+ beads using an optical-video method, as presented in Figure 12a–d. The configuration comprises a vibration-stabilized stereoscopic microscope with a digital camera and a specialized chamber to position the bead for undisturbed image focus during cross-linking. Based on this technique, a fast automatic protocol is provided for the characterization of alginate beads, such as calculating their volume, which can be correlated to their mechanical properties. Furthermore, Lin et al. [125] have observed the in situ shrinkage of alginate-based emulsion gel beads. It seems that the increase of Young′s modulus during gelation is combined with water loss and, consequently, shrinkage, leading to the formation of compact gelling structures. To conclude, the present section clarifies that alginate beads are produced mainly by extrusion-dripping methods, which can provide accurate visualizations of the formation process. Nevertheless, it is very challenging to characterize in situ the micro-structural conformations. The Table 1 presents cumulates the representative studies of Section 3 and Section 4 for the real-time recording of alginate gelation.
Table 1. Recent representative methods for the real-time recording of alginate in situ gelation.
Table 1. Recent representative methods for the real-time recording of alginate in situ gelation.
TechniqueExperimental DetailsResearch ObjectivesYear of
Publication		Reference
RheologyIn situ release of cross-linker on alginate/gelatin mixtureRheological investigation of gelation2009[63]
Incubation of alginate in internal cross-linker on rheometerIn situ evaluation of gelation behavior[66]
LED bottom rheometric plate for UV cross-linking gelationReal-time evaluation of mechanical properties2011[77]
Rheological characterization upon direct mixing of reactantsEvaluation of gelation speed2012[116]
In situ rheological characterization of fouled alginate membraneAssessment of the parameters manifesting alginate fouling layer2013[107]
Fluid hydrogel formation on rheometerControlling the shearing profile on gelation kinetics2014[68]
UV irradiation on rheometer for photo-activated ionic gelationMonitoring of the two-step gelation mechanism[78]
Rheological investigation upon ex situ mixing of reactantsRecording of gelation kinetics2015[69]
Petri dish with filter paper and a dialysis membrane on the rheometerRecording of alginate-Ca2+ kinetics2016[79]
Visible light-curable alginateControl of furfuryl alginate gel formation2020[76]
Rheometric setup for injection of Ca2+ into alginate through micro-holesDetailed recording of cross-linking gelation[82]
In situ micro-rheology of foulant membrane surfaceStudy on foulant mechanical response for effective cleaning2021[108]
CO2 in alginate solution to initiate the gelation on the rheometerGeneration and characterization of hydrogel foams2022[86]
MicrofluidicsMicrofabrication of alginate hydrogelsControlling of Ca2+ diffusion into alginate2010[99,100]
Microfluidic flow-focusing deviceSize and shape of the alginate-Ca2+ microparticles2013[117]
Visualization methodsVisualized extrusion-dripping methodPrediction models for alginate-Ca2+macrobeads2009[118]
Visualization of gelation on a petri dishDetermination of sol–gel transition and gelling time2011[101]
Photo correlation imaging during alginate gelationInvestigation of gelling kinetics formation2013[102]
Flow cell between two microscope slidesReal-time studying of gel front2016[103,104]
Microscopy on a drop-wise methodMicroscopical characterization of alginate-Ca2+ beads2017[119]
High-speed imaging system on drop impact gelationEffect of drop kinetic energy on gelation2018[120,121]
Custom setup for optical video microscopyMeasurement of alginate bead volume change over time2021[124]
Light-triggered methodsUV light in microchannelsProgress of UV-triggered gelation2010[98]
Capillary columnsCapillary formation of ionotropic alginate membranesPropose equation for gelation kinetics2013[106]
Cantilever sensorsDynamic-mode cantilever sensors on alginate gelationReal-time monitoring of gelation2020[87,88]
Texture analyzerCustomized bead formation on a texture analyzerIn situ mechanical texture evaluation of alginate beads2019[123]
SAXSSmall-angle X-ray scattering oncompetitive ligand exchangegelationDetermination of the local structure of hydrogels2019[92]
Time-induced methodsFTIR, Uv-vis, and Raman spectroscopy on alginate–acrylamide gelationIn situ mapping of gel structure2021[95]

5. Conclusions and Outlook

The present review highlights the significance of real-time cross-linking alginate in situ gelation recording. Demonstrating such advanced experimental methods provides an assessment of the reaction progress and the main factors evolved in each stage. Customized rheometric setups and microscopical and structural time-resolved techniques have been developed over the last several years to control cross-linking gelation. Therefore, both mechanical and morphological properties can be recorded during the formation of alginate hydrogels and beads. This allows researchers to observe the process as it occurs and to investigate the effects of concentration and the physicochemical properties of reactants on the gelation evolved in an initial fast stage, followed by a slower evolution. Additionally, based on real-time experimental data, kinetic models are developed to describe and adequately predict the cross-linking alginate reaction. After extensive analysis of the research studies, it is observed that the fast external gelation, in which the gel point appears in time scales of seconds, can be quantified in terms of its mechanical properties and gel front position. Therefore, with the present review, an in-depth comprehension of alginate gelation is provided for the purpose of optimizing the conditions of the cross-linking reactions.
The prospects for in situ gelation are encouraging, since it provides many advantages compared to conventional gelation procedures. The transition of the liquid reactants to gelling structures on the characterization location, e.g., the rheometric bottom plate and microscopical cell, provides precise tuning of the mechanical and morphological properties over an extended time. The present review constitutes a valuable guide for identifying the appropriate technique to study the dynamic response, not only of alginate gelation, but also of other complex systems. The advanced setups can be utilized to study in situ sensitive biological hydrogels without the presence of an increased temperature which may cause a degradation of the material. Additionally, the in situ characterization of other cross-linking polymers, such as pectin and polyurethane, can be achieved. For the abovementioned reasons, developing methods to control the mechanical and structural characteristics simultaneously is deemed imperative. This will lead to the synthesis of smart hydrogels for state-of-the-art applications.

Author Contributions

I.N.B.: Conceptualization, Writing—original draft preparation. T.B.G.: Conceptualization, Writing—review and editing. E.F.: Supervision, Conceptualization, Writing—review and editing. T.B.: Supervision, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. de Castro Spadari, C.C.; de Bastiani, F.W.M.S.; Lopes, L.B.; Ishida, K. Alginate nanoparticles as non-toxic delivery system for miltefosine in the treatment of candidiasis and cryptococcosis. Int. J. Nanomed. 2019, 14, 5187–5199. [Google Scholar] [CrossRef] [PubMed]
  2. Arroyo, B.J.; Bezerra, A.C.; Oliveira, L.L.; Arroyo, S.J.; de Melo, E.A.; Santos, A.M.P. Antimicrobial active edible coating of alginate and chitosan add ZnO nanoparticles applied in guavas (Psidium guajava L.). Food Chem. 2020, 309, 125566. [Google Scholar] [CrossRef] [PubMed]
  3. Lv, C.; Zhou, X.; Wang, P.; Li, J.; Wu, Z.; Jaio, Z.; Guo, M.; Wang, Z.; Wang, Y.; Wang, L.; et al. Biodegradable alginate-based sponge with antibacterial and shape memory properties for penetrating wound hemostasis. Compos. B Eng. 2022, 247, 110263. [Google Scholar] [CrossRef]
  4. Iravani, S.; Varma, R.S. Alginate-based micro- and nanosystems for targeted cancer therapy. Mar. Drugs 2022, 20, 598. [Google Scholar] [CrossRef]
  5. Puscaselu, R.G.; Lobiuc, A.; Dimian, M.; Covasa, M. Alginate: From food industry to biomedical applications and management of metabolic disorders. Polymers 2022, 12, 2417. [Google Scholar] [CrossRef]
  6. Wang, B.; Wan, Y.; Zheng, Y.; Lee, X.; Liu, T.; Yu, Z.; Huang, J.; Ok, Y.S.; Chen, J.; Gao, B. Alginate-based composites for environmental applications: A critical review. Crit. Rev. Environ. Sci. Technol. 2019, 49, 318–356. [Google Scholar] [CrossRef]
  7. Trica, B.; Delattre, C.; Gros, F.; Ursu, A.V.; Dobre, T.; Djelveh, G.; Michaud, P.; Oancea, F. Extraction and characterization of alginate from an edible brown seaweed (Cystoseira barbata) harvested in the Romanian Black Sea. Mar. Drugs 2019, 17, 405. [Google Scholar] [CrossRef]
  8. Chee, S.Y.; Wong, P.K.; Wong, C.L. Extraction and characterisation of alginate from brown seaweeds (Fucales, Phaeophyceae) collected from Port Dickson, Peninsular Malaysia. J. Appl. Phycol. 2011, 23, 191–196. [Google Scholar] [CrossRef]
  9. Sabra, W.; Zeng, A.P.; Deckwer, W.D. Bacterial alginate: Physiology, product quality and process aspects. Appl. Microbiol. Biotechnol. 2001, 56, 315–325. [Google Scholar] [CrossRef]
  10. Hay, I.D.; Rehman, Z.U.; Moradali, M.F.; Wang, Y.; Rehm, B.H.A. Microbial alginate production, modification and its applications. Microb. Biotechnol. 2013, 6, 637–650. [Google Scholar] [CrossRef]
  11. Draget, K.I.; Skjåk-Braek, G.; Smidsrød, O. Alginate based new materials. Int. J. Biol. Macromol. 1997, 21, 47–55. [Google Scholar] [CrossRef] [PubMed]
  12. Sun, J.; Tan, H. Alginate-based biomaterials for regenerative medicine applications. J. Mater. 2013, 6, 1285–1309. [Google Scholar] [CrossRef] [PubMed]
  13. Papajová, E.; Bujdoš, M.; Chorvát, D.; Stach, M.; Lacík, I. Method for preparation of planar alginate hydrogels by external gelling using an aerosol of gelling solution. Carbohydr. Polym. 2012, 90, 472–482. [Google Scholar] [CrossRef]
  14. Pham, L.H.P.; Ly, K.L.; Colon-Ascanio, M.; Ou, J.; Wang, H.; Lee, S.W.; Wang, Y.; Choy, J.S.; Phillips, K.S.; Luo, X. Dissolvable alginate hydrogel-based biofilm microreactors for antibiotic susceptibility assays. Biofilm 2023, 5, 100103. [Google Scholar] [CrossRef]
  15. Draget, K.I.; Skjåk-Bræk, G.; Stokke, B.T. Similarities and differences between alginic acid gels and ionically crosslinked alginate gels. Food Hydrocoll. 2006, 20, 170–175. [Google Scholar] [CrossRef]
  16. Ma, W.; Ling, S.; Zhang, J.; Chen, Z.; Xu, J. Microfluidic fabrication of calcium alginate helical microfibers for highly stretchable wound dressing. J. Polym. Sci. 2022, 60, 1741–1749. [Google Scholar] [CrossRef]
  17. Grant, G.T.; Morris, E.R.; Rees, D.A.; Smith, P.J.C.; Thom, D. Biological interactions between polysaccharides and divalent cations: The egg-box model. FEBS Lett. 1973, 32, 195–198. [Google Scholar] [CrossRef]
  18. Fang, Y.; Al-Assaf, S.; Phillips, G.O.; Nishinari, K.; Funami, T.; Williams, P.A.; Li, L. Multiple steps and critical behaviors of the binding of calcium to alginate. J. Phys. Chem. B 2007, 111, 2456–2462. [Google Scholar] [CrossRef]
  19. Supriya; Pal, C.K.; Sengupta, S.; Basu, J.K. One pot synthesis of nano Ag in calcium alginate beads and its catalytic application in p-Nitrophenol reduction with kinetic parameter estimation and model fitting. Can. J. Chem. Eng. 2021, 99, 359–373. [Google Scholar] [CrossRef]
  20. Merakchi, A.; Bettayeb, S.; Drouiche, N.; Adour, L.; Lounici, H. Cross-linking and modification of sodium alginate biopolymer for dye removal in aqueous solution. Polym. Bull. 2019, 76, 3535–3554. [Google Scholar] [CrossRef]
  21. Brus, J.; Urbanova, M.; Czernek, J.; Pavelkova, M.; Kubova, K.; Vyslouzil, J.; Abbrent, S.; Konefal, R.; Horský, J.; Vetchy, D.; et al. Structure and dynamics of alginate gels cross-linked by polyvalent ions probed via solid state NMR spectroscopy. Biomacromolecules 2017, 18, 2478–2488. [Google Scholar] [CrossRef]
  22. Song, E.H.; Shang, J.; Ratner, D.M. 9.08—Polysaccharides. Ιn Polymer Science: A Comprehensive Reference; Matyjaszewski, K., Möller, M., Eds.; Elsevier: Oxford, UK, 2012; Volume 9, pp. 137–155. [Google Scholar]
  23. Hecht, H.; Srebnik, S. Structural characterization of sodium alginate and calcium alginate. Biomacromolecules 2016, 17, 2160–2167. [Google Scholar] [CrossRef] [PubMed]
  24. Haug, A.; Smidsrød, O. Selectivity of some anionic polymers for divalent metal ions. Acta Chem. Scand. 1970, 24, 843–854. [Google Scholar] [CrossRef]
  25. Mørch, Ý.A.; Donati, I.; Strand, B.L.; Skjåk-Bræk, G. Effect of Ca2+, Ba2+, and Sr2+ on alginate microbeads. Biomacromolecules 2006, 7, 1471–1480. [Google Scholar] [CrossRef] [PubMed]
  26. Liu, X.; Qian, L.; Shu, T.; Tong, Z. Rheology characterization of sol–gel transition in aqueous alginate solutions induced by calcium cations through in situ release. Polymer 2003, 44, 407–412. [Google Scholar] [CrossRef]
  27. Lu, L.; Liu, X.; Qian, L.; Shu, T.; Tong, Z. Sol–gel transition in aqueous alginate solutions induced by cupric cations observed with viscoelasticity. Polym. J. 2003, 35, 804–809. [Google Scholar] [CrossRef]
  28. Huang, X.; Zhang, Y.; Li, F.; Zhang, J.; Dai, Y.; Shi, M.; Zhao, Y. Highly efficient alginate-based macromolecular photoinitiator for crosslinking and toughening gelatin hydrogels. J. Polym. Sci. 2020, 58, 1439–1449. [Google Scholar] [CrossRef]
  29. Pourjavadi, A.; Tavakolizadeh, M.; Hosseini, S.H.; Rabiee, N.; Bagherzadeh, M. Highly stretchable, self-adhesive, and self-healable double network hydrogel based on alginate/polyacrylamide with tunable mechanical properties. J. Polym. Sci. 2020, 58, 2062–2073. [Google Scholar] [CrossRef]
  30. Guo, B.; Ma, Z.; Pan, L.; Shi, Y. Properties of conductive polymer hydrogels and their application in sensors. J. Polym. Sci. B Polym. Phys. 2019, 57, 1606–1621. [Google Scholar] [CrossRef]
  31. Mei, X.; Ye, D.; Zhang, F.; Di, C. Implantable application of polymer-based biosensors. J. Polym. Sci. 2022, 60, 328–347. [Google Scholar] [CrossRef]
  32. Radebe, N.W.; Fengler, C.; Klein, C.O.; Figuli, R.; Wilhelm, M. A combined setup for correlating chemical changes via FTIR spectroscopy and rheological properties in a strain-controlled rheometer. J. Rheol. 2021, 65, 681–693. [Google Scholar] [CrossRef]
  33. Meng, Z.; Zhou, X.; Xu, J.; Han, X.; Dong, Z.; Wang, H.; Zhang, Y.; She, J.; Xu, L.; Wang, C.; et al. Light-triggered in situ gelation to enable robust photodynamic-immunotherapy by repeated stimulations. Adv. Mater. 2019, 31, 1900927. [Google Scholar] [CrossRef] [PubMed]
  34. Rhein-Knudsen, N.; Ale, M.T.; Ajalloueian, F.; Meyer, A.S. Characterization of alginates from Ghanaian brown seaweeds: Sargassum spp. and Padina spp. Food Hydrocoll. 2017, 71, 236–244. [Google Scholar] [CrossRef]
  35. Kopplin, G.; Lervik, A.; Draget, K.I.; Aachmann, F.L. Alginate gels crosslinked with chitosan oligomers—A systematic investigation into alginate block structure and chitosan oligomer interaction. RSC Adv. 2021, 11, 13780–13798. [Google Scholar] [CrossRef]
  36. Mikula, K.; Skrzypczak, D.; Ligas, B.; Witek-Krowiak, A. Preparation of hydrogel composites using Ca2+ and Cu2+ ions as crosslinking agents. SN Appl. Sci. 2019, 1, 643. [Google Scholar] [CrossRef]
  37. Milocco, A.; Scuor, N.; Lughi, V.; Lamberti, G.; Barba, A.A.; Divittorio, R.; Grassi, G.; Perkan, A.; Grassi, M.; Abrami, M. Thermal gelation modeling of a pluronic-alginate blend following coronary angioplasty. J. Appl. Polym. Sci. 2020, 137, 48539. [Google Scholar] [CrossRef]
  38. Urbanova, I.M.; Pavelkova, M.; Czernek, J.; Kubova, K.; Vyslouzil, J.; Pechova, A.; Molinkova, D.; Vyslouzil, J.; Vetchy, D.; Brus, J. Interaction pathways and structure−chemical transformations of alginate gels in physiological environments. Biomacromolecules 2019, 20, 4158–4170. [Google Scholar] [CrossRef]
  39. Jadach, B.; Świetlik, W.; Froelich, A. Sodium Alginate as a Pharmaceutical Excipient: Novel Applications of a Well-known Polymer. J. Pharm. Sci. 2022, 111, 1250–1261. [Google Scholar] [CrossRef]
  40. Zhao, J.; Li, B.; Liu, Z.; Dai, D.; Li, Y.; Shi, R.; Zhang, H. A novel solar-triggered MIL-125(Ti)/g-C3N4/SA composite aerogel with high catalytic activity for degradation of organic contaminants. Sep. Purif. Technol. 2021, 279, 119696. [Google Scholar] [CrossRef]
  41. Ul-Islam, M.; Ahmad, F.; Fatima, A.; Shah, N.; Yasir, S.; Ahmad, M.W.; Manan, S.; Ullah, M.W. Ex situ synthesis and characterization of high strength multipurpose bacterial cellulose-aloe vera hydrogels. Front. Bioeng. Biotechnol. 2021, 9, 601988. [Google Scholar] [CrossRef]
  42. Kuang, L.; Ma, X.; Ma, Y.; Yao, Y.; Tariq, M.; Yuan, Y.; Liu, C. Self-assembled injectable nanocomposite hydrogels coordinated by in situ generated CaP nanoparticles for bone regeneration. ACS Appl. Mater. Interfaces 2019, 11, 17234–17246. [Google Scholar] [CrossRef] [PubMed]
  43. Martin- Saldaña, S.; Al Waeel, M.; Alsharabasy, A.M.; Daly, A.; Pandit, A. An interdisciplinary framework for the characterization of extracellular matrix-hydrogels for biomedical applications. Matter 2022, 5, 3659–3705. [Google Scholar] [CrossRef]
  44. Holly, E.E.; Venkataraman, S.K.; Chambon, F.; Winter, H.H. Fourier transform mechanical spectroscopy of viscoelastic materials with transient structure. J. Nonnewton. Fluid Mech. 1988, 27, 17–26. [Google Scholar] [CrossRef]
  45. Fu, S.; Thacker, A.; Sperger, D.M.; Boni, R.L.; Buckner, I.S.; Velankar, S.; Munson, E.J.; Block, L.H. Relevance of rheological properties of sodium alginate in solution to calcium alginate gel properties. AAPS PharmSciTech 2011, 12, 453–460. [Google Scholar] [CrossRef] [PubMed]
  46. Puguan, J.M.C.; Yu, X.; Kim, H. Characterization of structure, physico-chemical properties and diffusion behavior of Ca-Alginate gel beads prepared by different gelation methods. J. Colloid Interface Sci. 2014, 432, 109–116. [Google Scholar] [CrossRef]
  47. Chan, L.W.; Lee, H.Y.; Heng, P.W.S. Mechanisms of external and internal gelation and their impact on the functions of alginate as a coat and delivery system. Carbohydr. Polym. 2006, 63, 176–187. [Google Scholar] [CrossRef]
  48. Kuo, C.K.; Ma, P.X. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: Part 1. Structure, gelation rate and mechanical properties. Biomaterials 2001, 22, 511–521. [Google Scholar] [CrossRef]
  49. Choi, B.Y.; Park, H.J.; Hwang, S.J.; Park, J.B. Preparation of alginate beads for floating drug delivery system: Effects of CO2 gas-forming agents. Int. J. Pharm. 2002, 239, 81–91. [Google Scholar] [CrossRef]
  50. Liu, X.D.; Yu, W.Y.; Zhang, Y.; Xue, W.M.; Yu, W.T.; Xiong, Y.; Ma, X.J.; Chen, Y.; Yuan, Q. Characterization of structure and diffusion behaviour of Ca-alginate beads prepared with external or internal calcium sources. J. Microencapsul. 2002, 16, 775–782. [Google Scholar] [CrossRef]
  51. Ramdhan, T.; Ching, S.H.; Prakash, S.; Bhandari, B. Time dependent gelling properties of cuboid alginate gels made by external gelation method: Effects of alginate-CaCl2 solution ratios and pH. Food Hydrocoll. 2019, 90, 232–240. [Google Scholar] [CrossRef]
  52. Kaklamani, G.; Cheneler, D.; Grover, L.M.; Adams, M.J.; Bowen, J. Mechanical properties of alginate hydrogels manufactured using external gelation. J. Mech. Behav. Biomed. Mater. 2014, 36, 135–142. [Google Scholar] [CrossRef]
  53. Liu, X.D.; Bao, D.C.; Xue, W.M.; Xiong, Y.; Yu, W.T.; Yu, X.J.; Ma, X.J.; Yuan, Q. Preparation of uniform calcium alginate gel beads by membrane emulsification coupled with internal gelation. J. Appl. Polym. Sci. 2003, 87, 848–852. [Google Scholar] [CrossRef]
  54. Sangroniz, L.; Fernández, M.; Santamaria, A. Polymers and rheology: A tale of give and take. Polymer 2023, 271, 125811. [Google Scholar] [CrossRef]
  55. Papageorgiou, M.; Kasapis, S.; Gothard, M.G. Structural and textural properties of calcium induced, hot-made alginate gels. Carbohydr. Polym. 1994, 24, 199–207. [Google Scholar]
  56. Dalheim, M.Ø.; Omtvedt, L.A.; Bjørge, I.M.; Akbarzadeh, A.; Mano, J.F.; Aachmann, F.L.; Strand, B.L. Mechanical properties of Ca-saturated hydrogels with functionalized alginate. Gels 2019, 5, 23. [Google Scholar] [CrossRef]
  57. Adibnia, V.; Hill, R.J. Universal aspects of hydrogel gelation kinetics, percolation and viscoelasticity from PA-hydrogel rheology. J. Rheol. 2016, 60, 541–548. [Google Scholar] [CrossRef]
  58. Sun, X.; Li, Z.; Cui, Z.; Wu, S.; Zhu, S.; Liang, Y.; Yang, X. Preparation and physicochemical properties of an injectable alginate-based hydrogel by the regulated release of divalent ions via the hydrolysis of D-glucono- δ-lactone. J. Biomater. Appl. 2020, 34, 891–901. [Google Scholar] [CrossRef]
  59. Kim, H.; Song, D.; Ngo, H.V.; Jin, G.; Park, C.; Park, J.-B.; Lee, B.-J. Modulation of the clinically accessible gelation time using glucono-d-lactone and pyridoxal 5′-phosphate for long-acting alginate in situ forming gel injectable. Carbohydr. Polym. 2021, 272, 118453. [Google Scholar] [CrossRef]
  60. Draget, K.I.; Skjåk Bræk, G.; Smidsrød, O. Alginic acid gels: The effect of alginate chemical composition and molecular weight. Carbohydr. Polym. 1994, 25, 31–38. [Google Scholar] [CrossRef]
  61. Nobile, M.R.; Pirozzi, V.; Somma, E.; Gomez D’Ayala, G.; Laurienzo, P. Development and rheological investigation of novel Alginate/N-Succinylchitosan hydrogels. J. Polym. Sci. B Polym. Phys. 2008, 46, 1167–1182. [Google Scholar] [CrossRef]
  62. Li, P.; Guo, C.; Li, X.; Yuan, K.; Yang, X.; Guo, Y.; Yang, X. Preparation and structural characteristics of composite alginate/casein emulsion gels: A microscopy and rheology study. Food Hydrocoll. 2021, 118, 106792. [Google Scholar] [CrossRef]
  63. Panouillé, M.; Larreta-Garde, V. Gelation behaviour of gelatin and alginate mixtures. Food Hydrocoll. 2009, 23, 1074–1080. [Google Scholar] [CrossRef]
  64. Li, A.; Gong, T.; Yang, X.; Guo, Y. Interpenetrating network gels with tunable physical properties: Glucono-δ-lactone induced gelation of mixed Alg/gellan sol systems. Int. J. Biol. Macromol. 2020, 151, 257–267. [Google Scholar] [CrossRef] [PubMed]
  65. Hu, C.; Lu, W.; Sun, C.; Zhao, Y.; Zhang, Y.; Fang, Y. Gelation behavior and mechanism of alginate with calcium: Dependence on monovalent counterions. Carbohydr. Polym. 2022, 294, 119788. [Google Scholar] [CrossRef]
  66. Funami, T.; Fang, Y.; Noda, S.; Ishihara, S.; Nakauma, M.; Draget, K.I.; Nishinari, K.; Phillips, G.O. Rheological properties of sodium alginate in an aqueous system during gelation in relation to supermolecular structures and Ca2+ binding. Food Hydrocoll. 2009, 23, 1746–1755. [Google Scholar] [CrossRef]
  67. Liao, H.; Ai, W.; Zhang, K.; Nakauma, M.; Funami, T.; Fang, Y.; Nishinari, K.; Draget, K.I.; Phillips, G.O. Mechanisms of oligoguluronate modulating the calcium-induced gelation of alginate. Polymer 2015, 74, 166–175. [Google Scholar] [CrossRef]
  68. Fernández Farrés, I.; Norton, I.T. Formation kinetics and rheology of alginate fluid gels produced by in-situ calcium release. Food Hydrocoll. 2014, 40, 76–84. [Google Scholar] [CrossRef]
  69. Larsen, B.E.; Bjørnstad, J.; Pettersen, E.O.; Tønnesen, H.H.; Melvik, J.E. Rheological characterization of an injectable alginate gel system. BMC Biotechnol. 2015, 15, 29. [Google Scholar] [CrossRef]
  70. Iatridi, Z.; Saravanou, S.-F.; Tsitsilianis, C. Injectable self-assembling hydrogel from alginate grafted by P(N-isopropylacrylamide-co-N-tert-butylacrylamide) random copolymers. Carbohydr. Polym. 2019, 219, 344–352. [Google Scholar] [CrossRef] [PubMed]
  71. Theodorakis, N.; Saravanou, S.-F.; Kouli, N.-P.; Iatridi, Z.; Tsitsilianis, C. pH/thermo-responsive grafted alginate-based SiO2 hybrid nanocarrier/hydrogel drug delivery systems. Polymers 2021, 13, 1228. [Google Scholar] [CrossRef]
  72. Goudoulas, T.B.; Germann, Ν. Phase transition kinetics and rheology of gelatin-alginate mixtures. Food Hydrocoll. 2017, 66, 49–60. [Google Scholar] [CrossRef]
  73. Li, X.; Weng, Y.; Kong, X.; Zhang, B.; Li, M.; Diao, K.; Zhang, Z.; Wang, X.; Chen, H. A covalently crosslinked polysaccharide hydrogel for potential applications in drug delivery and tissue engineering. J. Mater. Sci. Mater. Med. 2012, 23, 2857–2865. [Google Scholar] [CrossRef]
  74. Yang, J.; Steck, J.; Suo, Z. Gelation kinetics of alginate chains through covalent bonds. Extreme Mech. Lett. 2020, 40, 100898. [Google Scholar] [CrossRef]
  75. Wan, L.Q.; Jiang, J.; Arnold, D.E.; Guo, X.E.; Lu, H.H.; Mow, V.C. Calcium concentration effects on the mechanical and biochemical properties of chondrocyte-alginate constructs. Cell. Mol. Bioeng. 2008, 1, 93–102. [Google Scholar] [CrossRef]
  76. Heo, Y.; Akimoto, J.; Kobatake, E.; Yoshihiro, I. Gelation and release behavior of visible light-curable alginate. Polym. J. 2020, 52, 323–332. [Google Scholar] [CrossRef]
  77. Bonino, C.A.; Samorezov, J.E.; Jeon, O.; Alsberg, E.; Khan, S.A. Real-time in situ rheology of alginate hydrogel photocrosslinking. Soft Matter 2011, 7, 11510. [Google Scholar] [CrossRef]
  78. Higham, A.K.; Bonino, C.A.; Raghava, S.R.; Khan, S.A. Photo-activated ionic gelation of alginate hydrogel: Real-time rheological monitoring of the two-step crosslinking mechanism. Soft Matter 2014, 10, 4990–5002. [Google Scholar] [CrossRef]
  79. Mahdi, M.H.; Diryak, R.; Kontogiorgos, V.; Morris, G.A.; Smith, A.M. In situ rheological measurements of the external gelation of alginate. Food Hydrocoll. 2016, 55, 77–80. [Google Scholar] [CrossRef]
  80. Diryak, R.; Kontogiorgos, V.; Ghori, M.U.; Bills, P.; Tawfik, A.; Morris, G.A.; Smith, A.M. Behavior of in situ cross-linked hydrogels with rapid gelation kinetics on contact with physiological fluids. Macromol. Chem. Phys. 2018, 219, 1700584. [Google Scholar] [CrossRef]
  81. Senjoti, F.G.; Ghori, M.U.; Diryak, R.; Conway, B.R.; Morris, G.A.; Smith, A.M. Rheo-Dissolution: A new platform for the simultaneous measurement of rheology and drug release. Carbohydr. Polym. 2020, 229, 115541. [Google Scholar] [CrossRef] [PubMed]
  82. Besiri, I.N.; Goudoulas, T.B.; Germann, N. Custom-made rheological setup for in situ real-time fast alginate-Ca2+ gelation. Carbohydr. Polym. 2020, 246, 116615. [Google Scholar] [CrossRef]
  83. Besiri, I.N.; Goudoulas, T.B.; Germann, N. Impact of CaCl2 concentration and in situ rheometric setup configuration on fast alginate–Ca2+ reaction. Phys. Fluids 2022, 34, 053104. [Google Scholar] [CrossRef]
  84. Didonaki, A. Comparison of Hydrogels Using In Situ Characterization. Master’s Thesis, Technical University of Munich School of Life Sciences, Forschungsdepartment Life Science Engineering, Freising, Germany, 2022. [Google Scholar]
  85. Besiri, I.N.; Goudoulas, T.B.; Fattahi, E.; Becker, T. In situ evaluation of alginate-Ca2+ gelation kinetics. J. Appl. Polym. Sci. 2023, e54252. [Google Scholar] [CrossRef]
  86. Ben Djemaa, I.; Andrieux, S.; Auguste, S.; Jacomine, L.; Tarnowska, M.; Drenckhan-Andreatta, W. One-step generation of alginate-based hydrogel foams using CO2 for simultaneous foaming and gelation. Gels 2022, 8, 444. [Google Scholar] [CrossRef]
  87. Cesewski, E.; Singh, M.; Liu, Y.; Zhang, J.; Haring, A.P.; Johnson, B.N. Real-time monitoring of hydrogel rheological property changes and gelation processes using high-order modes of cantilever sensors. J. Appl. Phys. 2020, 128, 174502. [Google Scholar] [CrossRef]
  88. Haring, A.P.; Singh, M.; Koh, M.; Cesewski, E.; Dillard, D.A.; Kong, Z.J.; Johnson, B.N. Real-time characterization of hydrogel viscoelastic properties and sol-gel phase transitions using cantilever sensors. J. Rheol. 2020, 64, 837–850. [Google Scholar] [CrossRef]
  89. Sharma, H.; Lakshmanan, R.S.; Johnson, B.N.; Mutharasan, R. Piezoelectric cantilever sensors with asymmetric anchor exhibit picogram sensitivity in liquids. Sens. Actuators B Chem. 2011, 153, 64–70. [Google Scholar] [CrossRef]
  90. Ghernaouti, F.; Perrin, A.; Causse, J.; Chandre, F.; Cornu, D.; Cambedouzou, J. Small angle X-ray scattering to investigate the specific surface of hydrated alginate microbeads. Food Hydrocoll. 2022, 127, 107498. [Google Scholar] [CrossRef]
  91. Sikorski, P.; Mo, F.; Skjåk-Bræk, G.; Stokke, B.T. Evidence for egg-box-compatible interactions in calcium-alginate gels from fiber X-ray diffraction. Biomacromolecules 2007, 8, 2098–2103. [Google Scholar] [CrossRef]
  92. Yamamoto, K.; Yuguchi, Y.; Stokke, B.T.; Sikorski, P.; Bassett, D.C. Local structure of Ca2+ alginate hydrogels gelled via competitive ligand exchange and measured by small angle X-ray scattering. Gels 2019, 5, 3. [Google Scholar] [CrossRef]
  93. Bassett, D.C.; Håti, A.G.; Melø, T.B.; Stokkea, B.T.; Sikorski, P. Competitive ligand exchange of crosslinking ions for ionotropic hydrogel formation. J. Mater. Chem. B 2016, 4, 6175. [Google Scholar] [CrossRef]
  94. Håti, A.G.; Bassett, D.C.; Ribe, J.M.; Sikorski, P.; Weitz, D.A.; Stokke, B.T. Versatile, cell and chip friendly method to gel alginate in microfluidic devices. Lab Chip 2016, 16, 3718–3727. [Google Scholar] [CrossRef] [PubMed]
  95. Pragya, A.; Mutalik, S.; Younas, M.W.; Pang, S.-K.; So, P.-K.; Wang, F.; Zheng, Z.; Noor, N. Dynamic cross-linking of an alginate–acrylamide tough hydrogel system: Time-resolved in situ mapping of gel self-assembly. RSC Adv. 2021, 11, 10710. [Google Scholar] [CrossRef]
  96. LaGrow, A.P.; Lloyd, D.C.; Schebarchov, D.; Gai, P.L.; Boyes, E.D. In situ visualization of site-dependent reaction kinetics in shape-controlled nanoparticles: Corners vs edges. J. Phys. Chem. C 2019, 123, 14746–14753. [Google Scholar] [CrossRef]
  97. Potter, K.; Balcom, B.J.; Carpenter, T.A.; Hall, L.D. The gelation of sodium alginate with calcium ions studied by magnetic resonance imaging (MRI). Carbohydr. Res. 1994, 257, 117–126. [Google Scholar] [CrossRef]
  98. Chueh, B.; Zheng, Y.; Torisawa, Y.; Hsiao, A.Y.; Ge, C.; Hsiong, S.; Huebsch, N.; Franceschi, R.; Mooney, D.J.; Takayama, S. Patterning alginate hydrogels using light-directed release of caged calcium in a microfluidic device. Biomed. Microdevices 2010, 12, 145–151. [Google Scholar] [CrossRef]
  99. Braschler, T.; Valero, A.; Colella, L.; Pataky, K.; Brugger, J.; Renaud, P. Fluidic microstructuring of alginate hydrogels for the single cell niche. Lab Chip 2010, 10, 2771–2777. [Google Scholar] [CrossRef]
  100. Braschler, T.; Valero, A.; Colella, L.; Pataky, K.; Brugger, J.; Renaud, P. Link between alginate reaction front propagation and general reaction diffusion theory. Anal. Chem. 2011, 83, 2234–2242. [Google Scholar] [CrossRef]
  101. Nunamaker, E.A.; Otto, K.J.; Kipke, D.R. Investigation of the material properties of alginate for the development of hydrogel repair of dura mater. J. Mech. Behav. Biomed. Mater. 2011, 4, 16–33. [Google Scholar] [CrossRef]
  102. Secchi, E.; Roversi, Τ.; Buzzaccaro, S.; Piazza, L.; Piazza, R. Biopolymer gels with “physical” cross-links: Gelation kinetics, aging, heterogeneous dynamics, and macroscopic mechanical properties. Soft Matter 2013, 9, 3931. [Google Scholar] [CrossRef]
  103. Bjørnøy, S.H.; Bassett, D.C.; Ucar, S.; Andreassen, J.-P.; Sikorski, P. A correlative spatiotemporal microscale study of calcium phosphate formation and transformation within an alginate hydrogel matrix. Acta Biomater. 2016, 44, 254–266. [Google Scholar] [CrossRef] [PubMed]
  104. Bjørnøy, S.H.; Mandaric, S.; Bassett, D.C.; Åslund, A.K.O.; Ucar, S.; Andreassen, J.-P.; Strand, B.L.; Sikorski, P. Gelling kinetics and in situ mineralization of alginate hydrogels: A correlative spatiotemporal characterization toolbox. Acta Biomater. 2016, 15, 243–253. [Google Scholar] [CrossRef]
  105. Pfaff, N.-M.; Kleijn, J.M.; van Loosdrecht, M.C.M.; Kemperman, A.J.B. Formation and ripening of alginate-like exopolymer gel layers during and after membrane filtration. Water Res. 2021, 195, 116959. [Google Scholar] [CrossRef] [PubMed]
  106. Hassan, R.; Gobourib, A.; Zaafarany, I. Kinetics and mechanism of sol-gel transformation between sodium alginate anionic polyelectrolyte and some alkaline earth metal ions with formation of coordination biopolymer ionotropic polymembrane hydrogels of capillary structures. Adv. Biosens. Bioelectron. 2013, 2, 47–56. [Google Scholar]
  107. Sioutopoulos, D.C.; Goudoulas, T.B.; Kastrinakis, E.G.; Nychas, S.G.; Karabelas, A.J. Rheological and permeability characteristics of alginate fouling layers developing on reverse osmosis membraneσ during desalination. J. Membr. Sci. 2013, 434, 74–84. [Google Scholar] [CrossRef]
  108. Epstein, J.A.; Ramon, G.Z. In-situ micro-rheology of a foulant layer at a membrane surface. J. Membr. Sci. 2021, 640, 119747. [Google Scholar] [CrossRef]
  109. Gunatilake, U.B.; Venkatesan, M.; Basabe-Desmonts, L.; Benito-Lopez, F. Ex situ and in situ magnetic phase synthesised magneto-driven alginate beads. J. Colloid Interface Sci. 2022, 610, 741–750. [Google Scholar] [CrossRef]
  110. Chen, Z.; Chen, M.; Koh, K.Y.; Neo, W.; Ong, C.N.; Chen, J.P. An optimized CaO2-functionalized alginate bead for simultaneous and efficient removal of phosphorous and harmful cyanobacteria. Sci. Total Environ. 2022, 806, 150382. [Google Scholar] [CrossRef]
  111. Zhao, Y.; Carvajal, M.T.; Won, Y.-Y.; Harris, M.T. Preparation of calcium alginate microgel beads in an electrodispersion reactor using an internal source of calcium carbonate nanoparticles. Langmuir 2007, 23, 12489–12496. [Google Scholar] [CrossRef]
  112. Vicini, S.; Castellano, M.; Mauri, M.; Marsano, E. Gelling process for sodium alginate: New technical approach by using calcium rich micro-spheres. Carbohydr. Polym. 2015, 134, 767–774. [Google Scholar] [CrossRef]
  113. Blandino, A.; Macías, M.; Cantero, D. Formation of calcium alginate gel capsules: Influence of sodium alginate and CaCl2 concentration on gelation kinetics. J. Biosci. Bioeng. 1999, 88, 686–689. [Google Scholar] [CrossRef]
  114. Chan, A.W.; Whitney, R.A.; Neufeld, R.J. Kinetic controlled synthesis of pH-responsive network alginate. Biomacromolecules 2008, 9, 2536–2545. [Google Scholar] [CrossRef] [PubMed]
  115. Abdellaoui, Y.; Celaya, C.A.; Elhoudi, M.; Boualou, R.; Agalit, H.; Reina, M.; Gamero-Melo, P.; Abou Oualid, H. Understanding of vibrational and thermal behavior of bio-based doped alginate@nickel cross-linked beads: A combined experimental and theoretical study. J. Mol. Struct. 2022, 1249, 131524. [Google Scholar] [CrossRef]
  116. Lee, P.; Rogers, M.A. Effect of calcium source and exposure-time on basic caviar spherification using sodium alginate. Int. J. Gastron. Food Sci. 2012, 1, 96–100. [Google Scholar] [CrossRef]
  117. Dang, T.D.; Joo, S.W. Preparation of tadpole-shaped calcium alginate microparticles with sphericity control. Colloids Surf. B 2013, 102, 766–771. [Google Scholar] [CrossRef] [PubMed]
  118. Chan, E.-S.; Lee, B.-B.; Ravindra, P.; Poncelet, D. Prediction models for shape and size of ca-alginate macrobeads produced through extrusion–dripping method. J. Colloid Interface Sci. 2009, 338, 63–72. [Google Scholar] [CrossRef]
  119. Swioklo, S.; Ding, P.; Pacek, A.W.; Connon, C.J. Process parameters for the high-scale production of alginate-encapsulated stem cells for storage and distribution throughout the cell therapy supply chain. Process Biochem. 2017, 59, 289–296. [Google Scholar] [CrossRef]
  120. Haldar, K.; Chakraborty, S. Effect of liquid pool concentration on chemically reactive drop impact gelation process. J. Colloid Interface Sci. 2018, 528, 156–165. [Google Scholar] [CrossRef]
  121. Haldar, K.; Chakraborty, S. Role of chemical reaction and drag force during drop impact gelation process. Colloids Surf. A Physicochem. Eng. Asp. 2018, 559, 401–409. [Google Scholar] [CrossRef]
  122. Haldar, K.; Chakraborty, S. Investigation of chemical reaction during sodium alginate drop impact on calcium chloride film. Phys. Fluids 2019, 31, 072102. [Google Scholar] [CrossRef]
  123. Stößlein, S.; Grunwald, I.; Stelten, J.; Hartwig, A. In-situ determination of time-dependent alginate-hydrogel formation by mechanical texture analysis. Carbohydr. Polym. 2019, 205, 287–294. [Google Scholar] [CrossRef] [PubMed]
  124. Posbeyikian, A.; Tubert, E.; Bacigalupe, A.; Escobar, M.M.; Santagapita, P.R.; Amodeo, G.; Perullini, M. Evaluation of calcium alginate bead formation kinetics: An integrated analysis through light microscopy, rheology and microstructural SAXS. Carbohydr. Polym. 2021, 269, 118293. [Google Scholar] [CrossRef] [PubMed]
  125. Lin, D.; Kelly, A.L.; Maidannyk, V.; Miao, S. Effect of concentrations of alginate, soy protein isolate and sunflower oil on water loss, shrinkage, elastic and structural properties of alginate-based emulsion gel beads during gelation. Food Hydrocoll. 2020, 108, 105998. [Google Scholar] [CrossRef]
Polymers 15 02875 g003
Figure 3. Advantages of in situ gelation.
Figure 3. Advantages of in situ gelation.
Polymers 15 02875 g003
Polymers 15 02875 g004
Figure 4. (a) Aqueous solution of sodium alginate. (b) Aqueous solution consisting of insoluble strontium or calcium alginate particles. (c) Mixing of components. (d) Gelling ions migrate from strontium or calcium alginate particles to the alginate chains. (e) Formation of gel [69].
Figure 4. (a) Aqueous solution of sodium alginate. (b) Aqueous solution consisting of insoluble strontium or calcium alginate particles. (c) Mixing of components. (d) Gelling ions migrate from strontium or calcium alginate particles to the alginate chains. (e) Formation of gel [69].
Polymers 15 02875 g004
Polymers 15 02875 g005
Figure 5. (a) Modification of a lower rheometric plate on Malvern Gemini Nano HR rheometer. (b) Temporal evolution of G′ (open symbols) and G″ (filled symbols) of alginate–Ca2+ reaction [79]. Reprinted from Food Hydrocolloids, 55, M.H. Mahdi, R. Diryak, V. Kontogiorgos, G.A. Morris, A.M. Smith, “In situ rheological measurements of the external gelation of alginate”, 77–80, Copyright (2016), with permission from Elsevier.
Figure 5. (a) Modification of a lower rheometric plate on Malvern Gemini Nano HR rheometer. (b) Temporal evolution of G′ (open symbols) and G″ (filled symbols) of alginate–Ca2+ reaction [79]. Reprinted from Food Hydrocolloids, 55, M.H. Mahdi, R. Diryak, V. Kontogiorgos, G.A. Morris, A.M. Smith, “In situ rheological measurements of the external gelation of alginate”, 77–80, Copyright (2016), with permission from Elsevier.
Polymers 15 02875 g005
Polymers 15 02875 g006
Figure 6. (a) Customized rheometric setup for cross-linking reactions on an Anton Paar MCR 502 rheometer. (b) Detection of the sol–gel transition in an alginate–Ca2+ reaction [82]. Reprinted from Carbohydrate Polymers, 246, Besiri, I.N.; Goudoulas, T.B.; Germann, N., “Custom-made rheological setup for real-time fast alginate-Ca2+ in situ gelation”, 116615, Copyright (2020), with permission from Elsevier. (c) Improved setup and final configuration of Kinexus Ultra+ rheometer. (d) Kinetics at different angular frequencies when 1 wt.% alginate reacts with 100 mM Ca2+, and (e) long-term response of alginate-Ca2+ gelation, using the four micro-holes configuration [83]. Reprinted from Besiri, I.N.; Goudoulas, T.B.; Germann, N. “Impact of CaCl2 concentration and in situ rheometric setup configuration on fast alginate–Ca2+ reaction”. Phys. Fluids 2022, 34, 053104, with the permission of AIP Publishing.
Figure 6. (a) Customized rheometric setup for cross-linking reactions on an Anton Paar MCR 502 rheometer. (b) Detection of the sol–gel transition in an alginate–Ca2+ reaction [82]. Reprinted from Carbohydrate Polymers, 246, Besiri, I.N.; Goudoulas, T.B.; Germann, N., “Custom-made rheological setup for real-time fast alginate-Ca2+ in situ gelation”, 116615, Copyright (2020), with permission from Elsevier. (c) Improved setup and final configuration of Kinexus Ultra+ rheometer. (d) Kinetics at different angular frequencies when 1 wt.% alginate reacts with 100 mM Ca2+, and (e) long-term response of alginate-Ca2+ gelation, using the four micro-holes configuration [83]. Reprinted from Besiri, I.N.; Goudoulas, T.B.; Germann, N. “Impact of CaCl2 concentration and in situ rheometric setup configuration on fast alginate–Ca2+ reaction”. Phys. Fluids 2022, 34, 053104, with the permission of AIP Publishing.
Polymers 15 02875 g006
Polymers 15 02875 g007
Figure 7. Time sweeps of 1.2 mL 0.5 wt.% alginate—0.6 mL 25 mM CaCl2 using the setup of Besiri et al. [83]. Comparison of kinetics using alginates with different (a) Mw and (b) G/M [84]. Reproduced with permission from Didonaki, A., Comparison of hydrogels using in situ characterization. Master’s Thesis; published by Technical University of Munich School of Life Sciences, 2022.
Figure 7. Time sweeps of 1.2 mL 0.5 wt.% alginate—0.6 mL 25 mM CaCl2 using the setup of Besiri et al. [83]. Comparison of kinetics using alginates with different (a) Mw and (b) G/M [84]. Reproduced with permission from Didonaki, A., Comparison of hydrogels using in situ characterization. Master’s Thesis; published by Technical University of Munich School of Life Sciences, 2022.
Polymers 15 02875 g007
Polymers 15 02875 g008
Figure 8. (a) Graphical representation of the customized configuration for the time-dependent SAXS on alginate gelation. (b) Kratky plots during CLEX gelation of 1% alginate in aqueous MOPS buffer containing CaEDTA and ZnEDDA at Ca2+ = 30 mM and pH = 7.0. The data were recorded every 5 s for 145 s. Solid black lines indicate SAXS obtained from fully reacted samples following 10 h reaction time [92]. Reproduced with permission from Yamamoto et al., Gels; published by MDPI AG, 2019.
Figure 8. (a) Graphical representation of the customized configuration for the time-dependent SAXS on alginate gelation. (b) Kratky plots during CLEX gelation of 1% alginate in aqueous MOPS buffer containing CaEDTA and ZnEDDA at Ca2+ = 30 mM and pH = 7.0. The data were recorded every 5 s for 145 s. Solid black lines indicate SAXS obtained from fully reacted samples following 10 h reaction time [92]. Reproduced with permission from Yamamoto et al., Gels; published by MDPI AG, 2019.
Polymers 15 02875 g008
Polymers 15 02875 g009
Figure 9. (a) Flow cell for the alginate–Ca2+ gelation process. (b) Graphical representation of the calcium flux (blue arrows) in the gelled alginate (solid green color) (c) Temporal evolution of gel front as a function of the CaCl2 concentration. (d) Dark field (DF) microscopy images show the gel front position at different time intervals within the 100 mM CaCl2 flow in the system [104]. Reprinted from Acta Biomaterialia, 44, S.H. Bjørnøy, S. Mandaric, D.C. Bassett, A.K.O. Åslund, S. Ucar, J.-P. Andreassen, B.L. Strand, P. Sikorski, “Gelling kinetics and in situ mineralization of alginate hydrogels: A correlative spatiotemporal characterization toolbox”, 243–253, Copyright (2016); with permission from Elsevier.
Figure 9. (a) Flow cell for the alginate–Ca2+ gelation process. (b) Graphical representation of the calcium flux (blue arrows) in the gelled alginate (solid green color) (c) Temporal evolution of gel front as a function of the CaCl2 concentration. (d) Dark field (DF) microscopy images show the gel front position at different time intervals within the 100 mM CaCl2 flow in the system [104]. Reprinted from Acta Biomaterialia, 44, S.H. Bjørnøy, S. Mandaric, D.C. Bassett, A.K.O. Åslund, S. Ucar, J.-P. Andreassen, B.L. Strand, P. Sikorski, “Gelling kinetics and in situ mineralization of alginate hydrogels: A correlative spatiotemporal characterization toolbox”, 243–253, Copyright (2016); with permission from Elsevier.
Polymers 15 02875 g009
Polymers 15 02875 g010
Figure 10. (a) Schematic configuration for forming gel beads. (b) Successive images of crater dynamics and gel formation for drop impact from 15 mm [120]. Reprinted from Journal of Colloid and Interface Science, 528, K. Haldar, S. Chakraborty, “Effect of liquid pool concentration on chemically reactive drop impact gelation process”, 156–165, Copyright (2018), with permission from Elsevier.
Figure 10. (a) Schematic configuration for forming gel beads. (b) Successive images of crater dynamics and gel formation for drop impact from 15 mm [120]. Reprinted from Journal of Colloid and Interface Science, 528, K. Haldar, S. Chakraborty, “Effect of liquid pool concentration on chemically reactive drop impact gelation process”, 156–165, Copyright (2018), with permission from Elsevier.
Polymers 15 02875 g010
Polymers 15 02875 g011
Figure 11. (a) Experimental process for forming alginate beads. (b) Force of the pressurized beads at 1.4 mm to the sample holder as a function of the hydrogel formation time. The error bars correspond to the standard deviation of three experiments [123]. Reprinted from Carbohydrate Polymers, 205, S. Stößlein, I. Grunwald, J. Stelten, A. Hartwig, “In-situ determination of time-dependent alginate-hydrogel formation by mechanical texture analysis”, 287–294, Copyright (2019), with permission from Elsevier.
Figure 11. (a) Experimental process for forming alginate beads. (b) Force of the pressurized beads at 1.4 mm to the sample holder as a function of the hydrogel formation time. The error bars correspond to the standard deviation of three experiments [123]. Reprinted from Carbohydrate Polymers, 205, S. Stößlein, I. Grunwald, J. Stelten, A. Hartwig, “In-situ determination of time-dependent alginate-hydrogel formation by mechanical texture analysis”, 287–294, Copyright (2019), with permission from Elsevier.
Polymers 15 02875 g011
Polymers 15 02875 g012
Figure 12. (a) Microscopical configuration to measure the temporal bead volume change. (b) Two types of chambers are employed in the microscope. (c,d) Graphical representation describing the setup. [124]. Reprinted from Carbohydrate Polymers, 269, A. Posbeyikian, E. Tubert, A. Bacigalupe, M.M. Escobar, P. R. Santagapita, G. Amodeo, M. Perullini, “Evaluation of calcium alginate bead formation kinetics: An integrated analysis through light microscopy, rheology and microstructural SAXS”, 118293, Copyright (2021), with permission from Elsevier.
Figure 12. (a) Microscopical configuration to measure the temporal bead volume change. (b) Two types of chambers are employed in the microscope. (c,d) Graphical representation describing the setup. [124]. Reprinted from Carbohydrate Polymers, 269, A. Posbeyikian, E. Tubert, A. Bacigalupe, M.M. Escobar, P. R. Santagapita, G. Amodeo, M. Perullini, “Evaluation of calcium alginate bead formation kinetics: An integrated analysis through light microscopy, rheology and microstructural SAXS”, 118293, Copyright (2021), with permission from Elsevier.
Polymers 15 02875 g012
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Besiri, I.N.; Goudoulas, T.B.; Fattahi, E.; Becker, T. Experimental Advances in the Real-Time Recording of Cross-Linking Alginate In Situ Gelation: A Review. Polymers 2023, 15, 2875. https://doi.org/10.3390/polym15132875

AMA Style

Besiri IN, Goudoulas TB, Fattahi E, Becker T. Experimental Advances in the Real-Time Recording of Cross-Linking Alginate In Situ Gelation: A Review. Polymers. 2023; 15(13):2875. https://doi.org/10.3390/polym15132875

Chicago/Turabian Style

Besiri, Ioanna N., Thomas B. Goudoulas, Ehsan Fattahi, and Thomas Becker. 2023. "Experimental Advances in the Real-Time Recording of Cross-Linking Alginate In Situ Gelation: A Review" Polymers 15, no. 13: 2875. https://doi.org/10.3390/polym15132875

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop