Effects of Hydroxyapatite Additions on Alginate Gelation Kinetics During Cross-Linking

Abstract

Alginate hydrogels have gathered significant attention in biomedical engineering due to their remarkable biocompatibility, biodegradability, and ability to encapsulate cells and bioactive molecules, but much less has been reported on the kinetics of gelation. Scarce experimental data are available on cross-linked alginates (AL) with bioactive components. The present study addressed a novel method for defining the crosslinking mechanism using rheological measurements for aqueous mixtures of AL and calcium chloride (CaCl₂) with the presence of hydroxyapatite (HAp) as filler particles. The time-dependent crosslinking behaviour of these mixtures was exploited using a plate-plate rheometer, when crosslinking occurs due to calcium ions (Ca²⁺) binding to the guluronic acid blocks within the AL polymer, forming a stable "egg-box" structure. To reveal the influence of HAp particles as filler on crosslinked sample morphology, after rheological measurement and crosslinking, crosslinked samples were freeze-dried and their morphology was assessed using an optical microscope and SEM. It was found that the addition of HAp particles, which are known to enhance the mechanical properties and biocompatibility of crosslinked AL gels, significantly decreased (usually rapidly) the interaction between the Ca²⁺ and AL chains. In this research, the physical "shielding" effect of HAp particles on the crosslinking of AL with Ca²⁺ ions has been observed for the first time, and its crosslinking behaviour was defined using rheological methods. After crosslinking and rheometer measurements, the samples were further evaluated for morphological properties and the observations were correlated with their dewatering properties. While the presence of HAp particles led to a slower crosslinking process and a more uniform development of the rheological parameters, it also led to a more uniform porosity and improved dewatering properties. The observed effects allow for a better understanding of the crosslinking process kinetics, which directly affects the physical and chemical properties of the AL gels. The shielding behaviour (retardation) of filler particles occurs when they physically or chemically block certain components in a mixture, delaying their interaction with other reactants. In hydrogel formulations, filler particles like hydroxyapatite (HAp) can act as barriers, adsorbing onto reactive components or creating physical separation, which slows the reaction rate and allows for controlled gelation or delayed crosslinking. This delayed reactivity is beneficial for precise control over the reaction timing, enabling the better manipulation of material properties such as crosslinking distribution, pore structure, and mechanical stability. In this research, the physical shielding effect of HAp particles was observed through changes in rheological properties during crosslinking and was dependent on the HAp concentration. The addition of HAp also enabled more uniform porosity and improved dewatering properties. The observed effects allow for a better understanding of the crosslinking process kinetics, which directly affects the physical and chemical properties of the AL gels.

Keywords: calcium alginate hydrogels; crosslinking kinetics; hydroxyapatite; rheology.

Figure 1

Schematic presentation of crosslinking of polyguloronate sequences of the alginate (AL) chain by a dimerization mechanism with Ca²⁺ ions via formation of the “egg-box” structure. Formed porous scaffolds made of crosslinked hydrogels enable bodily fluids transport through their matrix and can be used for tissue engineering and bone regeneration [5,14].

Figure 2

SEM images of hydroxyapatite (HAp) particles used in this research at two different magnifications, revealing particles of micrometre dimensions with a spherical structure (a,b).

Figure 3

A schematic overview of the experimental setup in this study includes the preparation of suspensions, the molecular structure and formation of the egg-box network, rheological measurements leading to crosslinked structure formation, followed by SEM imaging and dewatering measurements.

Figure 4

Schematic presentation of experimental set-up prior to rheological measurements; samples are mixed on the bottom plate of the rheometer prior to initiation of the measurements, leading to crosslinking of the AL solution with crosslinking dispersion of the suspension.

Figure 5

Schematic presentation of the experimental set up used for the gravimetric water retention device ÅA-GWR; crosslinked samples were placed between blotter papers and a porous membrane, influencing water retention.

Figure 6

Viscoelastic properties of alginate (AL) and calcium chloride (CaCl₂) crosslinking samples as a function of strain amplitude for constant angular frequency 0.1 rad·s⁻¹ and increase in concentrations of HAp (a) 0.3% CaCl₂, (b) 0.4% CaCl₂ and (c) Calcium cloride CaCl₂. Effect of concentration increase of CaCL₂ and HAp on development of viscoelasticity and increase of elastic moduli as the crossover point where G′ = G″.

Figure 7

Frequency sweep results indicating reaction dynamic and crosslinking kinetics within the angular frequency (ω) range 0.01–150 rad·s⁻¹ (a) for crosslinking suspensions containing both CaCl₂ and HAp particles and (b) for crosslinking solutions containing only Ca²⁺ ions.

Figure 8

Elastic (G′) and loss (G″) moduli dependence on two constant strain (γ) values of 0.01% (a,b) and 0.1% (c,d) and constant angular frequencies (ω) of 0.1, 1, 2.5, 5 and 10 rad·s⁻¹ for samples crosslinked with 0.3% CaCl2 (a,c) and 0.4% CaCl2 (b,d). Storage modulus (G′) full lines, loss modulus (G″) open lines.

Figure 9

Viscoelastic moduli (G′ and G″)values for different constant strain values (γ = 0.01% (a,b) and γ = 0. 1% (c,d)) and different angular frequencies (a) G′ for concentration 0.3% CaCl₂, (b) G″ for concentration 0.3% CaCl₂, (c) G′ for concentration 0.4% CaCl₂ and (d) G″ for concentration 0.4% CaCl₂.

Figure 10

Crosslinked cluster formation during junction of calcium ions with alginate molecular chains presented with time-dependent increase of transient complex viscosity (η^*+) increase up to the final crosslinked structure when the plateau value is reached (a) as a function of the concentration of CaCl₂ and the presence of HAp particles, (b) gelation influenced only by the presence of Ca²⁺ ions and (c) maximum of transient complex viscosity η^*+ _max.

Figure 11

Structure formation during time dependent fluid gel crosslinking of alginate gels with calcium ions revealed with an increase in static stress (τ_s). (a) Rheograms of static stress during crosslinking and (b) average values of maximum of reached static stress(τ_smax), revealing the effect on overshoot due to instant monocomplex formation, containing compact swollen structures when suspended without HAp particles. An increase in calcium ion concentration and HAp particle concentration increases the crosslinking time.

Figure 12

Optical camera images of the alginate samples after crosslinking as a function of calcium ion concentration and the amount of HAp filler particles. An increase of HAp filler particles increases the opacity and colour of the samples, becoming white with fewer large pores.

Figure 13

SEM images of samples after gelation. The difference in structure between more open layered pores with a lower amount of CaCl₂ concentration, from 0.3 CaCl₂ to 0.4% CaCl₂, and more particles of Hap attached to alginate chains for 20% to 60% HAp, as revealed by a rheometer.

Figure 14

Correlation among gels with different concentration of CaCl₂. Yield stress and dewatering properties of crosslinked samples as a function of the amount of HAp particles, using a ÅA GWR device for evaluation of the porosity of the crosslinked samples. The presence of HAp particles improves water retention and leads to a more uniform porosity, facilitating better liquid distribution through the samples.