Larsen et al.

BMC Biotechnology (2015) 15:29
DOI 10.1186/s12896-015-0147-7

RESEARCH ARTICLE Open Access

Rheological characterization of an injectable
alginate gel system
Benjamin Endré Larsen1*, Jorunn Bjørnstad3,4, Erik Olai Pettersen2, Hanne Hjorth Tønnesen1 and Jan Egil Melvik3,5

Abstract
Background: This work investigates a general method for producing alginate gel matrices using an internal mode
of gelation that depends solely on soluble alginate and alginate/gelling ion particles. The method involves the
formulation of two-component kits comprised of soluble alginate and insoluble alginate/gelling ion particles.
Gelling kinetics, elastic and Young’s moduli were investigated for selected parameters with regard to soluble
alginate guluronate content, molecular weight, calcium or strontium gelling ions and alginate gelling ion particle
sizes in the range between 25 and 125 micrometers.
Results: By mixing the two components and varying the parameters mentioned above, alginate gel matrices with
tailor-made viscoelastic properties and gelling kinetics were obtained. Final gel elasticity depended on alginate type,
concentration and gelling ion. The gelling rate could be manipulated, e.g. through selection of the alginate type
and molecular weight, particle sizes and the concentration of non-gelling ions.
Conclusions: Formulations of the injectable and moldable alginate system presented have recently been used
within specific medical applications and may have potential within regenerative medicine or other fields.
Keywords: Alginate gel, Biocompatible, Gelling kinetics, Gel system, Rheological characterization

Background bioartificially engineer nerve tissue [12-14], investigate
Hydrogels in the form of cross-linked hydrophilic poly- and treat cartilage defects [15,16] and various other
mers that adsorb large amounts of water without dissol- tissues [17,18].
ution are widely used as growth matrices and scaffolds Alginate hydrogels are most frequently produced by
in regenerative medicine, as well as in numerous other exposing an aqueous solution of alginate polymer to
biomedical applications [1-6]. Alginates have interesting calcium ions, but many other di- and multivalent metal
properties in this respect. They are biopolymers which cations may also be used. As an alternative to Ca2+, both
can be extracted from macroalgae or bacterial cultures Ba2+ and Sr2+ produce stronger, yet still biocompatible
and consist of unbranched binary copolymers of 1–4 alginate gels [19-22]. Gelling occurs when cations coord-
glycosidically linked α-L-guluronic acid (G) and its C-5 inate with the charged substituents of the monomer moi-
epimer β-D-mannuronic acid (M) [7]. eties, leading to the formation of interchain complexes
Both preclinical and clinical studies have shown that involving several guluronate moieties on adjacent alginate
alginates are highly biocompatible [8,9]. Because of their strands. Given sufficient concentration of alginate and
ability to form gels under physiologically relevant condi- binding ions, this gives rise to a three-dimensional net-
tions, they are widely used and studied for encapsulation work in the form of a gel. The binding zone between the
purposes and as biostructure materials. Entrapment of G-blocks is often described by the so-called “egg-box
cells in alginate beads is a commonly used technique model” [23]. More recent research, however, has shown
[10,11], and alginates have been shown to be a useful that other gel formation mechanisms involving alternating
and promising material for other types of biostructures. (MG) structures also play a significant role [24,25].
Among other applications, they have been used to Alginate hydrogels are commonly formed by the dialy-
sis/diffusion method, where an alginate solution is gelled
* Correspondence: benjamil@gmail.com by diffusion of gelling ions from an outer reservoir. This
1
School of Pharmacy, University of Oslo, Oslo, Norway
Full list of author information is available at the end of the article method is the most frequently used when making alginate

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Alginate gel systems which are formulated with the in. Sev. Also. It is therefore often less vironment while preserving a high degree of viability. a rapid process limited by the diffusion of tem allows conveniently entrapping cells in a 3D en- gelling ions into the gel network. The manufacturing of alginate microbe. Commonly. liquid which would be sufficiently non-viscous to be A high content of guluronic acid resulted in a storage injected and thereafter solidify to a firm gel matrix. ions. as a function of time is shown for both to diffuse into the alginate solution to give a calcium alginates. and that it was useful in a model system for the de- This is both because the rapid gelling process limits the termination of cell respiration in this respect [34.35]. In such systems. and It has also been useful in constructs for cartilage re- because this mode of gelation frequently leads to an in. G’. the gel made using high-mannuronate sodium alginate ate gel formulations with a chemistry that is compatible gelled with high-guluronate strontium alginate. Results Alternative methods for manufacturing biocompatible The gel system studied (Figure 1) allowed variation of alginate gel structures also exist. A detailed descrip- forms. as measured by Young’s modulus.physiological pH values have obvious alginate dispersion had similar characteristics as that of limitations.31]. be timed or otherwise regulated. measured along its radius [22]. The alginate particle dispersion could be modi- calcium salt with limited solubility. for several purposes. in order to increase the legibility of the figure as it showed tent to delay the gelling process could be advantageous a similar tendency. Importantly. compared calcium alginate particles were also investigated (Figure 2A). The rate of gel forma. to diffusion systems. tan(G”/G’). We have here studied sev.e. molecular weight and concentra- Here. formulations were also subjected to compression tests The present method. properties. while loss modulus. where analyses showed that gels formed in suspension of internally cross-linked alginate particles the absence of exogenous substances had similar elastic with sodium alginate solution. systems where gelation takes dium alginate gelled with high-mannuronate calcium place under non. generation and a formulation is currently undergoing homogeneous gel [27].26]. several parameters including the guluronate fraction of tion may be reduced by using internal gelling systems. Also. arc- saturated gel. but differed in conditions have been used in order to form a controllable their composition with respect to the guluronic to man- alginate gelling system [32. they differed with respect to maximal compressibility. Release the rheometer the first measurement could be recorded of calcium ions and gelling kinetics may also be con. and after the viscous gel- been used as a cross-linking agent in alginate based cell ling mass was ejected onto the serrated holding plate of delivery vehicles for tissue engineering [28-30]. as the concentration of alginate clinical trials as a treatment for dilated cardiomyop- in the shaped structure produced will correspond to athy [36-38]. to allow nutes and the storage modulus subsequently showed a the addition of cells or other biomaterials to a carrier stabilizing tendency as a result of the sol–gel transition. In Figure 2B the development trolled by using calcium salts with pH-dependent of storage moduli over time is shown for high-guluronate solubility and the addition of a slowly acting acid such as strontium alginate particle dispersions in combination D-glucono-δ-lactone [18. alginate gel nuronic acid ratio. The sodium calcium ions and designed to rupture under specific alginates had similar molecular weight. the soluble alginate. as eral parameters influencing gelling kinetics and elasticity measured by true strain at maximal corrected stress. modulus that was almost three times greater than for eral formulations of alginate gel systems may yet have the formulation with lower guluronic acid content in the important limitations. useful in the production of other shapes or structures. however. time available to mold the gel structure to shape. constitutes such an alter.33]. where calcium ions are allowed Storage modulus. These with a wider range of biomedical applications. and the rate of gelation may be controlled by tion of the alginate components used can be found in modifying the chemistry of the release. Calcium sulfate has ges took less than 5 seconds. although native alginate gelling system. . the gelation could. are shown only for the high guluronate alginate. The gel formed using high-guluronate so- the gelling kinetics. Thereafter. Depending on the applications case of gels formed using strontium alginate dispersions there may be problems associated with the control of (Figure 2B). the gelling ions are released inside the gel as it tions of the formulation constituents. liposomes loaded with with two different sodium alginate solutions. G”. are mixed with an alginate solution. the distance to the source reservoir. Formulations made using the same systems based upon internal gelling may have a more sodium alginates and a dispersion of high-mannuronate defined and limited supply of gelling ions. We have earlier found that the gel sys- ads is. a Table 1. BMC Biotechnology (2015) 15:29 Page 2 of 12 gel beads [22. It may therefore be useful to develop algin. a Mixing the components using the two connected syrin- release of calcium ions is triggered.Larsen et al. or complexed Ca2+ fied with respect to particle size and gelling ion species. in particular. and phase angle i. which is based on a mixture of a (Table 2). Gel formation occurred rapidly during the first mi- for instance. within about 90 seconds.

65 Sr 130 kDA Table 2 1.0% 0.0% 0.69 257 kDa 1.47 Ca 176 kDa The given concentrations are for the final gel mixture. A reciprocal migration of non-gelling ions associated with the soluble alginate also takes place.44 222 kDa 1. . and insoluble strontium or calcium alginate particles dispersed in an aqueous medium (B).1% 0.0% 0.69 219 kDa 1.47 Ca 176 kDa 1.1% 0.1% 0.70 222 kDa 1.1% 0.0% 0.65 Sr 130 kDA Figure 5 0.0% 0. The individual components reside in each of two syringes connected with a three-way connector (C). Table 1 Alginate gelling formulations tested Figure Sodium alginate Ca/Sr alginate Concentration FG MW Concentration FG Bound ion MW Figure 2 (lower panel) 1.69 219 kDa 0.4-1.45 220 kDa 1.4-1. FG is the guluronate fraction.65 Sr 130 kDA Figure 4 1.0% 0.1% 0.70 220 kDa 1.6% 0. The system consists of an aqueous sodium alginate solution (A).65 Sr 130 kDA 1.65 Sr 130 kDA 2.47 Ca 176 kDa Figure 6 1.1% 0.69 70 kDa 1.4-1.6% 0.0% 0.45 222 kDa 1.70 219 kDa 1.65 Sr 130 kDA 0.0% 0.65 Sr 130 kDA 1.69 70 kDa 1.1% 0.6% 0.69 219 kDa 1.0% 0. gelling ions migrate from the strontium or calcium alginate particles (D).0% 0.47 Ca 176 kDa 1. Upon mixing.0% 0.0% 0.69 219 kDa 0.0% 0. MW is the molecular weight.4-1. BMC Biotechnology (2015) 15:29 Page 3 of 12 Figure 1 Schematic illustration of the two-component alginate system and the method of gel formation.47 Ca 176 kDa Figure 3 1.Larsen et al. The gelling ions coordinate with binding sites on two adjacent chains (E) and a gel is formed.0% 0.65 Sr 130 kDA Figure 2 (upper panel) 1.0% 0.6% 0.

3 min G' .0 vs.0. BMC Biotechnology (2015) 15:29 Page 4 of 12 3500 12 Calcium 3000 Storage and loss modulus (G''.2 +/. The self-gel of 257 kDa to 70 kDa and used in the gel formulation. The total alginate salt concentration was 2.2 min 1000 0 0 10 20 30 40 50 60 Time after mixing (min) Figure 2 Oscillatory rheological studies of two different sodium alginates.0.0 +/.4 +/. 2.0. Calculated maximum elasticity (A) and half time (t1/2) for the gels are shown. 1.0.Larsen et al. centration on the gel formation.1 kPa 10 t=1.0. (3. Storage (G’) and loss (G”) moduli.7 with MW = 219 kDa.0% (w/w) i.e. the properties of the resulting gels were concentration in the formulation from 1.0.45 % guluronate 3000 A=2.70 % guluronate 4000 G' . formulations were gelled with the same solution of so- The data shown in Figure 3 demonstrate both a higher dium alginate (Figure 4).95 +/. An alginate batch with The effect of different particle size distributions was a high content of guluronic acid was degraded by auto.70 % guluronate 6 500 δ .1 +/.0.44 with MW = 222 kDa) gelled using calcium (upper) and strontium (lower) alginate particles.70 % guluronate 200 100 4 0 8000 Strontium A=8.1%.1 min 70 % Guluronate: 2000 A=3. whereas larger .03 kPa 2000 t1/2= 2. All data points are shown as the mean with standard error of at least three independent runs and curves were fitted to the data by eq.0. G') [Pa] 45 % Guluronate: 2500 A=3. 2. quite different: Smaller particles led to a higher rate of gel- sulted in an approximately two-fold increase in the storage ation and a lower final storage modulus.0% from each of the two components (upper panel). The studies were conducted on two different alginates (Fg = 0.0% to 2.1% from strontium alginate (lower panel).0% from sodium alginate and 1. Although the concentrations of gelling rate for the light alginate and that the gel stabilized both alginate components in the formulations tested were at lower elasticity values. as well as phase angle (tan (G”/G’)) as a function of time after mixing are shown for gels made of sodium alginate containing ~45% and 70% guluronic acid mixed with calcium alginate.1 min Storage modulus (G') [Pa] 6000 5000 G' . studied by subjecting the same calcium alginate batch to claving the alginate solution [39] from a molecular weight milling and sifting using a range of sieves. consisting of 1.70 % guluronate 8 1500 G' .2 kPa 7000 t1/2= 5. Fg = 0.3 +/.6 minutes). 1.0% re.6 +/.3 kPa Phase angle (δ) (°) t=2.45 % guluronate 1000 G'' . but did not significantly change the gelling rate the influence of the molecular weight and alginate con.1 +/. A further experiment was performed in order to study modulus. Increasing the soluble alginate always equal.

because of the high gelling rate. 1.15 +/. ~70% guluronate.4 59. Ultimate stress was calculated as the upper inflection point of the stress–strain curve. independently of the alginate concentration.1 kPa Increasing the fraction of sodium alginate above this level 5000 t1/2= 2. particle size resulted in lower gelling rates and higher gel gelling ion-alginate dispersion was also further explored elasticity. For these 3000 observations.0. The gelling rate increased strongly in alginate salt concentration was 2. 2 and 3. respectively). consisting of 1. formed where the relative content of the components some mechanical degradation of the gel probably occurred were varied while keeping the total alginate concentration while the two components were still being displaced constant at 2. All data points are shown as the mean constant.0. Gels of similar composition (containing ~45% and ~70% guluronate in the sodium alginate component) but treated differently.u) 0.5 ml 2% sodium alginate solution with a guluronate content of ~45% or ~70% and 1. ~45% guluronate.04 0. show a statistically significant difference at the p < 0. maximal stress was statistically different from the others at p < 0.61% w/w NaCl for 2 hrs. BMC Biotechnology (2015) 15:29 Page 5 of 12 Table 2 Compression tests on gels with different guluronate content with or without treatment with additional calcium ~45% guluronate.0%.double also resulted in a lower final storage modulus. For untreated gels. There was no such difference for treated gels (p = 0. rather than an abrupt break.0.5 hrs and subsequently incubated in 0. In Figure 6. with the number of parallels given in the first row. the inflection point. and the half time and storage modulus of the sodium alginate solution and final gel elasticity were plotted against the fraction of sodium alginate in the formulations. but was in concentration this case followed by a reduction in the gel formation rate. Calculated maximum gel strength (A) and half time due to rapid gelling before the gel/solution was placed (t1/2) for the gel setting are shown.92 ± 0. Gels were either left in the mold for approx. The curves were ob- 12000 tained both for calcium and strontium alginate particles of A=10. similarly to the formulations . The reduced gel elasticity observed at 0.5 ml of 2% calcium alginate dispersion.001 level (based on corrected readings). Young’s modulus was calculated as the initial linear slope of the resultant stress–strain plot.15.0.39 ± 0.3 min shorter when using strontium alginate as the gelling ion Storage Modulus (Pa) 8000 donor.0% from sodium alginate and 1.3 +/. The total chloride is shown.7) on formulations with different concentrations of sodium with different MW were combined with strontium alginate.9 +/.3 kPa the same sizes (45–75 μm). maximum gel elasti- 7000 city values were obtained when the soluble alginate frac- 6000 tion was kept at about 30% of the total alginate content. was the top of a plateau. Engineering stress (σeng) and strain (εeng) values were converted to corrected stress (σc) and Hencky’s strain (εh) according to eqs. the strain at maximal stress was statistically different at the p < 10−7 level for both corrected and uncorrected measurements.4 min as gelling rate seemed to follow a similar pattern for both 10000 types of particles although the final storage modulus was 9000 several times higher and gelation half time generally A=8. In the case of Ca2+-treated gels.02 kPa t1/2= 5. from each formulation (not shown) were fitted by non- The relative contribution to gelation half time and final linear regression as described above.0% (total 0.01 1. however. the gelling rate seemed to remain MW=257 kDa constant.4 ± 0.1%. under the rheometer probe. For each formulation and treatment. Guluronate-rich sodium alginates (Fg = 0. For the smallest particle sizes (less than 25 μm) (Figure 5): A series of oscillation experiments were per- it should be noted that. At the concentrations tested.2 9.005.8 85 ± 4 Hencky strain at maximal stress (εh.6 +/. no treatment no treatment Ca-treated Ca-treated Number of tests (n) 6 8 4 6 Young’s modulus (E.0 ± 0. For concentrations up to where the sodium alginate concentration was increased to 2.1% from strontium alginate except for the upper curve the presence of sodium ions.3 min resulted in reduced gel elasticity. There is no statistically significant difference between the Young’s moduli of gels having received the same treatment.37 and p = 0. All shear stress-time curves obtained between the syringes. obviously as a result of a 4000 reduction in the gelling ion concentration.0 +/. 0 0 10 20 30 40 50 60 The gelling process was found to be strongly influ- Time after mixing (min) enced by the presence of non-gelling ions.62 ± 0. kPa) 6. kPa) 20 ± 3 21 ± 1 90 ± 10 120 ± 10 Maximal stress (σc.0.0.5% NaCl the final gel elasticity was found to be relatively alginate concentration was 3.09 Compression tests were performed on gels made from 1. 3 hrs or left in the mold for 1.8 ± 0. a Figure 3 Storage modulus and kinetics of alginate gels as a function of series of oscillatory shear stress measurements performed time and molecular weight.1%).Larsen et al.u.9% NaCl with standard error of three independent runs and curves were fitted to was likely due to the fact that the gel was partly disrupted the data by eq. A=5. ~70% guluronate.55% w/w CaCl2 and 0.08 ± 0.01 0. 2000 MW=70 kDa Decreasing the sodium alginate concentration below 30% 1000 MW=70 kDa .0 +/. All values are given as mean ± standard error. The final gel elasticity as well 11000 t1/2= 3.01 and p < 0.

The curves were fitted by eq.0% from sodium alginate and 1. Upper panel: Storage modulus of alginate gels as a function of time for gels made of sodium alginate (Fg = 0.Storage modulus 4 10000 Storage modulus (Pa) Half time (min) 3 8000 6000 2 4000 1 2000 0 0 < 25 25-45 45-75 75-125 > 125 Particle size (µm) Figure 4 Storage modulus and kinetics of alginate gels as a function of particle size.Larsen et al.7 and MW = 219 kDa) and Sr alginate at different particle sizes. The total alginate concentration was 2. consisting of 1. hexametaphosphate.1%. . The data shown in source (data not shown). having the smallest particle size. and are shown when exceeding the dimension of point markers. Not surprisingly. Lower panel: Calculated maximum storage modulus (A) and half time (t1/2) for the fitted data. A similar dependency binding compound. 1 to the average data from three independent runs. Error bars denote the standard error of the mean calculated at each data point.1% from Sr alginate. and this was also was also found when using calcium alginate as gelling ion found to increase the gelling rate. BMC Biotechnology (2015) 15:29 Page 6 of 12 14000 13000 12000 11000 Storage modulus (Pa) 10000 9000 8000 7000 6000 5000 4000 < 25 µm 75-125 µm 3000 25-45 µm >125 µm 2000 45-75 µm 1000 0 0 5 10 15 20 25 30 35 40 Time after mixing (min) 5 14000 t1/2 12000 A . the final Figure 6 were obtained from gels made using strontium gel strength was lowered in the presence of a strontium alginate as the gelling ion source.

5 0. It has also been demonstrated that certain ranges of elasticity in hydrogel Discussion cell carriers may have a cytoprotective effect [49]. > 3. components to allow administration through commonly strated by the elasticity build up and the phase angle used syringes and to be used in combination with cells. For calcium and strontium based gels ions was significantly stronger for guluronate-rich gels.4 0.7 and MW = 219 kDa) though there are no established limits for the viscosity of in a mixture with Sr alginate or Ca alginate. which the final gel. It is well known that alginates with a higher the optimal range with respect to the magnitude of G’ content of guluronic acid may give higher gel strength. Al- function of the fraction of sodium alginate (Fg = 0. [22]. A reduced gelling rate may therefore be ob- 0. Final storage modulus (A) and half the use of higher concentrations of high MW alginates in time (t1/2) calculated from fitted oscillatory rheology curves. lead to a higher rate of calcium cated that the sol–gel transition occurred already before ion release into the gel-forming mix. Figure 5 Storage modulus of alginate gels as a function of alginate fraction in solution or particulate. and may be a result of differences in . BMC Biotechnology (2015) 15:29 Page 7 of 12 a large difference for the strontium based gels (Figure 2B).e. very high (i.6 0. to a lower gelation half time and lower elastic modulus in ling from an external reservoir commonly used. due to the increase in surface area The low phase angle observed in our experiments indi. per total particle weight. The sol–gel transition component (Figure 4) led to gelation responses which of alginates is regulated by the availability of free gelling were mainly a function of the calcium release kinetics: ions and has previously been studied in details [40-43]. while more than tripling the molecular weight 18 of the sodium alginate component led to an increase in Half time (min) 16 14 elastic modulus with a concomitant increase in gelling 12 half time. The The new alginate gelling system developed here is based formulations described here clearly have sufficiently low upon the exchange of gelling ions between soluble and viscosity during the first minutes after mixing the two insoluble alginate fractions. while longer gelation times allow for the of gel forming ions like Ca2+ and Sr2+ which was tested evolution of more optimal cross-linking configurations. The latter. for applica- tions using the current gel system for cell administration. too viscous to be practically useful [48]. is the result of will saturate the gel with gelling ions [44]. Gel formation was demon. the current in rapidly setting gels being stabilized at non-optimal thermo- situ gel formulations is limited with respect to availability dynamic minima. and loss modulus time series confirmed the rapid sol– Increasing the particle size of the calcium alginate gel phase transition (Figure 2A). The MW dependency may be interpreted as the 6 4 result of faster availability of shorter chains during gelling 2 while the formed gel may suffer with less entanglement or 0 MW overlap. was about 30%.8 Fraction sodium alginate tained by using high MW alginates.0%.45]. 20 However.0%) concen- concentration was always 2. 2000 Higher molecular weight alginate chains resulted in a 0 higher elastic modulus in the gels that were formed.7 0. Smaller particles will. The limitation in gelling ions will have effects on the The overall effect of varying the relative amount of the gel system which is clearly demonstrated by the change two components on the storage modulus appears similar in strength and elasticity by adding extra gelling ions for both gelling ions (Figure 5).3 0. This in turn leads the start of the measurements. and was found to be in (Table 2).Larsen et al. and gelling half time when the sodium alginate fraction but this may not be the case at lower gelling ion concen. Bars demonstrating standard trations of sodium alginate might yield solutions that are error of the mean are indicated when exceeding the symbols. 10000 The effects may be due to differences in flexibility between Storage modulus (Pa) Ca the two alginate types and the difference in gel forming 8000 Sr properties between calcium and strontium [24] The lack 6000 of difference in elasticity when using calcium alginate particles was also demonstrated by the calculated Young’s 4000 modulus. as a particular may be limited by the high viscosity [46]. although the data differed significantly with respect to compressive strength (Table 2). The difference between the two gelling trations [25. The total alginate injectable preparations [47]. However.2 0. a there also seemed to be a difference in gelling kinetics result that is in agreement with previous works by and elastic behavior between the two alginate types with Mørch et al.1 0. doubling its concentration nearly doubled the 10 elastic modulus with no significant change in the gelling 8 kinetic. as 22 did an increase in alginate concentration (Figure 3). it can be speculated. In contrast to dialysis gel. here.

7 and MW = 219 kDa) and Sr alginate in the presence or absence of sodium chloride or hexametaphosphate. Oscillatory stress-time curves were fitted to the data as described to obtain final storage modulus (A) and half time (t1/2). BMC Biotechnology (2015) 15:29 Page 8 of 12 10000 9000 8000 7000 Storage modulus (Pa) 6000 5000 4000 3000 Water 0.1%.5 % NaCl 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Time after mixing (min) 6 11000 10000 t1/2 5 9000 Storage modulus (Pa) A 8000 Half time (min) 4 7000 6000 3 5000 4000 2 3000 1 2000 1000 0 0 Water 0.0% from sodium alginate and 1. for the effect of the soluble alginate concentration.1% from Sr alginate. gelling the gelling mix increases the rate of ion exchange with . The total alginate concentration was 2. consisting of 1.9% NaCl 0.Larsen et al. Lower panel: Final storage modulus and half time (with standard error). Upper panel: Storage modulus of alginate gels as a function of time for gels made of sodium alginate (Fg = 0. All data points are shown as the mean with standard error of three independent runs (only one run for 0. when the sodium The rate of gelation was strongly increased by the alginate concentration was reduced below about 30% of addition of sodium ions to the sodium alginate solution the total alginate content of a formulation (amounting (Figure 6).9 % NaCl 2000 0.5% NaCl 0. calculated from fitted curves.4% Hexa- 0.met. residues on the alginate chains [24. The presence of an excess of sodium ions in to 2% w/w of the aqueous two-component system).1 % NaCl metaphosphate 1000 0.25]. The data also As the particles were found to remain insoluble and stable demonstrated that the use of calcium alginate particles for several months in aqueous solutions (data not shown) gave a higher gelling rate compared to similar strontium this might be the result of approaching a threshold level alginate particles (Figure 5). the mode of complex formation with the uronic acid half time was increased for both particle types (Figure 5). ence in binding mechanisms.1% NaCl 0. Figure 6 Storage modulus and kinetics of alginate gels as a function of non-gelling salts.phos. which may reflect the differ. However.4% H.9% NaCl).

a system that allows the formulation be useful for injection.55]. its molecular weight. alginate concentration mannitol or glucose. similar to what was previously discussed for cell cultivation. Conclusion tion of 0. was determined by NMR spectroscopy. age modulus and gelling kinetics. thus the chemical complexity is re. and may be avoided as gelling ions. At sufficiently models. the ratio of guluronate to mannuronate in by using a secondary. Dispersions of calcium and strontium alginates to regulate by varying the formulation parameters. smaller calcium alginate particles (Figure 4). Final particle size tion could not be used to fit our data. A first-order equa. neutral tonicity regulator such as the alginate. This resulted in clear and viscous solu- 3D cultures [54. alginate samples were of high guluronic acid content The results of the measurements of storage modulus (~70%). alginates (FMC Biopolymers/NovaMatrix. Becton Dickinson here is currently being tested in preclinical and clinical Infusion Therapy AB). It can be speculated determined by size-exclusion chromatography with light that the dual kinetic behavior may result from ion release scattering detection (given as weight average) and the taking place both from the particle surface – an effect that sequence distribution of guluronic and mannuronic acid is related to particle size – and from the particle interior.Larsen et al. is – within a certain range – possible tions. like within cell and biofactor administration. ally fragmented and washed repeatedly in water to remove component kinetic equation used could be well fitted to excess Ca or Sr ions before drying and the final product all measured elasticity data (although not for wider par. BMC Biotechnology (2015) 15:29 Page 9 of 12 the calcium donor due to entropic forcing. The tage with the present method compared to many other solution of soluble alginate and calcium or strontium alternatives may be that alginate and gelling ions are the alginate dispersion was conveniently mixed by using two only materials used. tissue bulking and tissue engineering. and particle distribution were shown to influence the stor- The addition of a calcium binding agent. tions (1–2%) with calcium or strontium salts solutions This is supported by the very quick gelling that occurs before freeze-drying and milling. was measured to contain approximately stoichiometric ticle distributions. The two. This together with the tunable properties of the high concentrations of sodium chloride this might have gel system demonstrated here therefore indicates potential led to gel stabilization at more entropic thermodynamic other uses. but has previously was controlled by an adapted milling and sieving pro- been found useful for glucono-delta-lactone initiated re. Norway). The results demonstrated that an isotonic sodium chloride concentra. All strontium as particles unfold. as passage through a syringe will of injectable alginate gels with an internal mode of gel- partly destroy the gel network. Variations in structure elasticity which has been freeze-dried alginate and stirring or shaking the vials for shown to influence differentiation and function of cells in at least one hour. amounts of Ca (~10%) or Sr (~20%). Furthermore. Details on the alginates used in each experiment as a function of time correspond well with previously can be found in Table 1. The observed increase in the rate of gelation strengthens this view. as thermo.52]. were prepared before use as the alginate particles was Due to the high biocompatibility of alginates an advan. This has been seen in other ation was presented. The contents of the two syringes . insoluble in water in the absence of soluble alginate. minima. The two-component al- ametaphosphate. published work on the elasticity of alginate gels [51. the effects of an increase in the number of Gel preparations cross-links were similar to those in gels produced using Solutions of sodium alginates at appropriate concentra- a concentration-dependent internal mode of gelation based tions were made by adding a corresponding amount of on hydrophobic association of modified alginate amides solvent to vials containing a standardized amount of [53]. Methods dynamic equilibrium would be obtained more rapidly Alginates in a calcium-limited gel. calcium present in the system. sodium hex.9% might result in a formulation which may not In the present work. cedure. The gels were mechanic- in contact with alginate and free gelling ions. The molecular weight of alginate samples was lease of Ca2+ ions from CaCO3 [45]. The amount added was to be a biocompatible and versatile matrix for use in sufficient to bind a large portion of the total amount of regenerative medicine and other biomedical applications. results not shown). resulted in a marked reduction in final ginate system presented produces injectable and moldable storage modulus due to the overall constraint in the gel matrices of a material that several studies have shown availability of calcium (Figure 6). syringes connected with a Luer lock connection and duced. In- Our interpretation of the data presented here is that soluble calcium and strontium alginates were prepared the most important factor influencing the gelling rate from sodium alginate by precipitation of alginate solu- was the rate of gelling ion release from the particles. As mentioned earlier the gel technology presented using a manifold (Connecta Plus 3. The effects of calcium and strontium two-component systems as well [50]. because of a reduction in the All samples tested were made from PRONOVA sodium number of possible coordination sites.

drafted the manuscript and performed the the curves in Figure 3. was head of R&D at this company when the presented studies were strength. The threshold of statistical significance was set at p < 0. Norway. the resultant mixture was displaced Excess saline solution was carefully wiped off and the back and forth between the syringes nine times before the specimens were transferred to the Texture Analyzer’s gelling mix was emptied through the manifold’s third sample container. NY/ Stable Micro Systems.Larsen et al. Northampton. and the Statistical analyses and data processing temperature was kept at 20°C during the experiments. Gels were prepared as described was found as the slope of a linear regression over the above and deposited directly onto the serrated base of data part recorded at the engineering strain interval of the rheometer.e. A is final gel the technology studied in the present work. An S/N4 cylindrical flat-tip probe with channel into an appropriate container and allowed to a cross-section of 4 mm was used. programmed to measure force while the probe traversed med in a rehearsed manner and timed to a maximum of the 6 mm gap between the probe’s initial position and 5 seconds. Redmond. alginate solutions and insoluble alginate dispersions steril. lel measurements. serrated probe.. which were transferred to 6-well culture plates containing an aqueous solution with 0. t is time. UK). US-MA). using at least three paral- data could be well fitted to equation (1). University of Oslo. except for out the compression tests. corrected stress using equations (2) and (3). The probe was subsequently lowered and 0 – 0. alginate dispersion. 5Current address: as molds. The set-up εh ¼ − ln 1 − εeng ð2Þ of the experiments was timed to 60 seconds from the  first syringe-to-syringe displacement. EOP and HHT participated in the design of the study and (OriginLab. Norway.005). After Author details 1 mixing the gel components between syringes. frequency: the procedure used by Mao et al.5 hrs. Scarsdale. Oslo. 3FMC Biopolymer AS. and markets products based on them. The other authors do not have any competing interests. Curve fittings and drawings were performed by using Authors’ contributions BEL and JB carried out the oscillatory rheology measurements. JEM conceived of the study and helped draft the manuscript. MA). Norway. σ c ¼ σ eng 1 − εeng ð3Þ The strain setting was chosen based on strain sweep experiments determining the viscoelastic window of the gels under investigation (results not shown). Oslo. [39]. convert position data to en- Oscillatory rheological measurements gineering strain and force readings to pressure. Horley.  G0 ¼ A 1−f ⋅e−k 1 t −ð1−f Þe−k 2 t ð1Þ Competing interests FMC Biopolymer AS holds several patents relating to alginate technologies. gap: 1 mm. Goren A. Norway. Goren E. 2009. mass was transferred to 24-well plates. some samples were carefully extracted from the wells Received: 18 July 2014 Accepted: 21 April 2015 using a flat spatula and cut to 5 mm thick slices. contribution fraction between the two exponential parts.XT2 Texture The authors thank FMC Biopolymer AS. Natick.55% w/w CaCl2 (50 mM) and References 1. Godalming. . The instrument was form a gel (Figure 1).22 μm filtered the sample container surface at a speed of 3 mm/min. low-viscosity silicone oil was applied at the outer edges  in order to prevent evaporation from the gel. Baruch L. The mesenchymal stem cells: a unique hypoimmunogenic platform for remaining samples were kept in the mold for the same long-term cellular therapy.0 (Erithacus Software. duration of time. according to ing system: PP50. up to 5% compression. 4Current address: Elopak AS. A custom script was used to remove head space readings.05. Sandvika. BEL carried GraFit 5. 2Department of Physics. Origomar AS. Oslo. k1 and k2 are rate constants and f is a relative conducted. for its kind provision of samples and access to laboratories. unless otherwise detailed. FASEB J. After this period. The data The setting of the gel with time (storage modulus) was were in turn converted to Hencky’s (true) strain and measured using a Physica MCR 300 rheometer (Measur. The displacement itself was perfor. One of the authors.0 statistical analyses. Mathworks. the gel School of Pharmacy. Sandvika. All statistical analyses were performed in Excel 2010 In all cases it was empirically found that the rheological (Microsoft. Machluf M. Encapsulated human 0. University of Oslo. BMC Biotechnology (2015) 15:29 Page 10 of 12 were mixed by first displacing the sodium alginate solu. and left to set for 1. Some of these are directly relevant to where G’ is storage modulus. Compression tests Acknowledgements Fully set gels were examined using a TA. Surrey.24(1):22–31. and were subsequently extracted in the tion into the syringe containing the strontium or calcium same manner and cut to 5 mm thick slices. UK). All authors read and approved the final manuscript.05. i. US-WA). helped revise the manuscript. Raw data were imported to MATLAB® R2010a (The ized by autoclaving were used. Analyzer (Texture Technologies Corp. Young’s modulus 1 Hz and strain: 0. which were used Norway. Spikkestad.61% w/w NaCl and left to incubate for 3 hrs. Jan Egil Melvik. For sterile gel preparations 0. Dahan N. Norway. which were drawn in Origin 8.

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