4B20 Laboratory- Crosslinking of Alginate for Biomaterial Applications 1
Abstract—Alginate has been used extensively in the biomedical industry for years. This paper examines the biomedical
applications of ionic and covalent crosslinking of alginate. Ionic
gelation of alginate beads and microspheres for cell culture is
carried out successfully. Comparisons between external and internal ionic crosslinking are made and it is shown that internal
crosslinking is better suited to injection into the human body due
to its lower gelation rate. The effects of alginate concentration in a
solution are examined and it is found that an increase in
concentration leads to better functional properties up to a certain extent. Shape memory properties of covalently cross-linked
alginate porous scaffolds and their biomedical applications are
explored. Ionic and covalent crosslinking of alginate provides
extensive biomaterial applications and the author believes further
research into its uses would be positive for the biomedical industry.
Keywords —Alginate, Cross-linking, Drug delivery, Gelation
I. INTRODUCTION
Alginate is used for various biomedical applications due to
its biocompatibility, relatively low cost, low toxicity, and mild
gelation (through divalent cations). Some of these applications
include wound healing [1], drug delivery [2], and tissue
engineering [3]. This paper aims to explore various methods of
cross-linking alginate and examine its mechanical properties
and stability through five different experiments. These are
introduced and explained below.
A. The Ionic Gelation of Alginate Beads for Cell Culture
Ionic gelation can be used to create alginate beads which are
used in biomedical applications. Ionic gelation is the simplest
method of gelation. A cross-linking agent (in our case,
calcium chloride) is used to form cross -links in alginate. In
other words, our polyelectrolyte solution (Na+ + alginate-) is
gelled with a multivalent ion of opposite charge (Ca2+ + 2Cl-).
[4]
B. The Ionic Gelation of Alginate Microspheres for Cell
Culture
Electrohydrodynamic spraying (electro-spray) is a method
used to disperse liquid. Microspheres are formed using
electro-spray that can be used in a range of applications such
as: stem cell carriers [5], in enzyme immobilizat ion [6], and
Chirag Chadha is a student in the B.A.I. and M.A.I. Biomedical Engineering
course at Trinity College, Dublin (phone: 353-83 106 3787; email:
for drug delivery [7]. Electrospray is used to create alginate
microspheres of varying sizes for cell culture using the two
solutions from A.
C. Comparing External and Internal Ionic Crosslinking
Alginate can undergo ionic crosslinking through external
gelation or internal gelation. In this section, the applications
and reasons for use of both of these crosslinking methods are
examined.
D. Effect of Alginate Concentration (0.5%, 1%, and 2%) on
Functional Properties
Using a Zwick machine, the functional properties of samples
with varying alginate concentrations are tested. The
equilibrium modulus and dynamic modulus of each sample is
calculated using results outputted by the Zwick machine.
E. Shape Memory Properties of Covalently Cross-linked
Alginate Porous Scaffolds
Covalent cross-linking is a permanent form of bonding that
is used to create alginate porous scaffolds. The shape memory
properties of covalently cross-linked alginate are explored.
Ionic and Covalent Crosslinking of Alginate for Biomaterial Applications
Chirag Chadha, Trinity College Dublin
Figure 1. Zwick machine setup with sample
4B20 Laboratory- Crosslinking of Alginate for Biomaterial Applications 2
This paper intends to concisely convey and educate the
reader on the biomedical applications and properties of ionic
and covalent cross-linking of alginate.
II. METHODS
A. The Ionic Gelation of Alginate Beads for Cell Culture
In order to produce ionic gelation of alginate beads for cell
culture to occur, it is necessary to first create a 100mM calcium
chloride (CaCl2) solution. To make 1 litre of solution, add
100ml of 1M CaCl2 to 900ml of ultra-pure water (UPW). Weigh
out alginic acid powder for the alginate solution and add UPW.
A 1% weight/volume (w/v) ratio requires 100mg of alginic acid
powder mixed with 10ml of UPW. Vortex this mixture and
rotate for 3 hours at 10RPM, or until it is fully dissolved.
Once the solutions are ready, pour 10ml of the CaCl2 solution
into a petri dish. Using a syringe and needle, slowly drop
alginate solution into the CaCl2 solution. If done properly,
alginate beads should form. Slowly swirl the petri dish if the
beads are not readily apparent.
B. The Ionic Gelation of Alginate Microspheres for Cell
Culture
Through the use of electrohydrodynamic spraying (electro-
spray) technology, microspheres were fabricated for cell
culture. Alginate concentration, pressure, and working distance
remained constant throughout the fabrication but applied
voltage and needle gauge were varied. The following table
summarizes the constants and variables.
Table 1. Electro-spray constants and variables.
C. Comparing External and Internal Ionic Crosslinking
External Gelation
As in A, add UPW to alginic
acid powder at a 1% w/v ratio. It
is necessary to use a mold for this
experiment in order to create an
alginate slab. Inject the alginate
solution into the mold and
immerse the mold into the
100mM CaCl2 solution from
before. Allow the alginate to set
and then punch out 5mm cores
using a biopsy punch.
Internal Gelation
A dual syringe is necessary for this section. Pipette alginate
solution into one syringe and calcium sulfate (CaSO4) slurry
into the other. Push the mixture from the dual syringe through
the mold and allow 30 minutes to set. As before, use a biopsy
punch to make 5mm cores.
D. Effect of Alginate Concentration (0.5%, 1%, and 2%) on
Functional Properties
Results from the unconfined alginate mech tests conducted on
the Zwick machine are used in this section. The Zwick machine
outputs force (N), time (s), stress (N/mm2), and strain (m) for
the equilibrium modulus tests and force (N), time (s), and strain
(m) for the dynamic modulus tests. Note that the Zwick
machine outputs strain as a change in length. This needs to be
divided by the height of the sample to obtain the actual value of
strain.
Equilibrium modulus (kPa)
=
Average force equilibrium (N)Cross sectional area (m2)⁄
Applied strain (10% )1000
⁄
=
Average force equilibrium (N)𝜋𝑟2⁄
0.11000⁄
Dynamic modulus (kPa)
=
Average force/time amplitude (N)Cross sectional area (m2)⁄
Average strain/time amplitude1000
⁄
Figure 2. Mold used to form
alginate slab
Figure 3. Force-time plot for 2% equilibrium test
0
0.01
0.02
0.03
0.04
0.05
0 500 1000 1500 2000 2500
4B20 Laboratory- Crosslinking of Alginate for Biomaterial Applications 3
=
Average force/time amplitude (N)𝜋𝑟2⁄
Average strain/time amplitude1000
⁄
Repeat these calculations for three sets of data and obtain
average results for equilibrium and dynamic modulus at each
alginate concentration.
III. RESULTS
A. The Ionic Gelation of Alginate Beads for Cell Culture
As the alginate solution is
introduced to the calcium chloride
solution through a syringe and
needle, ionic gelation will occur and
alginate beads will form. The result
of the ionic gelation is presented in
Figure 6. The beads can be seen as
small white circles in the petri dish.
B. The Ionic Gelation of Alginate Microspheres for Cell
Culture
The results of electro-spray on the alginate solution are
shown below.
It is evident that increasing the voltage applied upon the
solution decreases the sizes of the microspheres produced.
Also, comparing the two pictures, it is clear that the smaller
the needle (note: 26G < 21G), the finer the beads. Therefore,
the two variables, voltage applied and needle gauge, have
significant effects on the microsphere size.
C. Comparing External and Internal Ionic Crosslinking
External Gelation
Through external gelation, we obtain a 5mm core that is
shown in Figure 7.
Internal Gelation
Mixing the calcium sulfate slurry with alginate solution
creates a mixture that is much more liquid in form. Therefore,
we cannot use the biopsy punch in this case. See Figure 8.
Figure 6. Alginate beads in
petri dish.
Figure 4. Force-time plot for 2% dynamic test
0
0.005
0.01
0.015
0.02
0 5 10 15
Figure 5. Strain-time plot for 2% dynamic test
-0.005
0
0.005
0.01
0.015
0 5 10 15
Figure 7. 21G needle electro-spray
results
Figure 8. 26G needle electro-
spray results
Figure 7. External gelation
Figure 8. Internal gelation
4B20 Laboratory- Crosslinking of Alginate for Biomaterial Applications 4
D. Effect of Alginate Concentration (0.5%, 1%, and 2%) on
Functional Properties
Equilibrium modulus over 0.5%, 1%, and 2% concentrations
Dynamic modulus over 0.5%, 1%, and 2% concentrations
E. Shape Memory Properties of Covalently Cross-linked
Alginate Porous Scaffolds
The covalently cross-linked alginate porous scaffold
available in the lab displayed shape memory properties. The
sample could be compressed and deformed. This was done
with a lab sample spoon. Pouring PBS (saline) over the sample
made it swell up to its original size, thus displaying shape
memory properties.
IV. DISCUSSION
A. The Ionic Gelation of Alginate Beads for Cell Culture
The size and shape of the alginate beads were largely
dependent upon working distance. A larger working distance
created larger beads, while a smaller working distance created
smaller beads. In other words, the sooner a bead drops, the
smaller it will be. In this experiment, a sharp needle was used.
However, the shape of the bead would change if a blunt needle
were used. Similarly, the gauge of the needle would also affect
the size of the bead. It also would be possible to cause the
bead to drop sooner by increasing the viscosity of the solution,
leading to a smaller bead. [9]
It would not be possible to inject alginate beads as they are
simply too large. However, drug delivery through oral
ingestion could be possible. The beads could also be used to
treat external ailments through wound dressings.
B. The Ionic Gelation of Alginate Microspheres for Cell
Culture
As you squeeze the plunger of the syringe, the alginate
solution remains on the tip of the needle for a small amount of
time due to surface tension. Applying a voltage to the alginate
solution will cause the surface tension to negate leading to a
much quicker release. This results in a microsphere that is far
smaller than any bead that could be formed in A. A higher
voltage will negate the surface tension even sooner. This is
why the microspheres obtained through higher voltage electro-
spray were much smaller than their lower voltage
counterparts. It should be noted that bead size will only
decrease down to a certain level through electro-spray. [10]
Although alginate beads cannot be injected into the human
body, alginate microspheres could easily be injected due to
their size. They can be used for cell encapsulation, giving a
protective effect to whatever they carry. Cell encapsulation
would also result in delayed release time and could allow
localized drug delivery in the body. [5]-[7]
C. Comparing External and Internal Ionic Crosslinking
External gelation is easier to induce but can cause gelation to
occur too quickly, resulting in high solubility in aqueous
solutions. External gelation is known for being inhomogenous.
[8]-[11]
Internal gelation slows gelation rate through the use of
calcium sulfate (CaSO4) or calcium carbonate (CaCO3) which
have lower solubilities than CaCl2 allowing more time for
injection of the solution into the body. Internal gelation can
also work in bursts, meaning that any carriers will be released
sporadically. This could be advantageous or disadvantageous
depending upon application. [8]-[11]
D. Effect of Alginate Concentration (0.5%, 1%, and 2%) on
Functional Properties
It is evident from both graphs that the increase in alginate
concentration in our sample leads to an increase in functional
properties. More extensive studies have found similar results
[12]. The slight decrease in dynamic modulus from the 0.5%
alginate concentration to the 1% concentration could be
attributed to error in the Zwick machine, variation of sample,
error in setting up the experiment etc.
E. Shape Memory Properties of Covalently Cross-linked
Alginate Porous Scaffolds
The shape memory properties of covalently cross -linked
alginate porous scaffolds could be a huge benefit in the
biomedical field. They are biocompatible, non-toxic, and non-
mutagenic in the human body. They could revolutionize drug
delivery. However, degradation of chemical cross -linking
releases toxic chemicals which are harmful to the body. [13]-
[14]
y = 2.7126x + 0.2127R² = 0.9998
0
1
2
3
4
5
6
0 0.5 1 1.5 2 2.5
Equ
libri
um
mo
du
lus
Alginate Concentration (%)
y = 7.5873x + 0.2688R² = 0.8932
0
5
10
15
20
0 0.5 1 1.5 2 2.5
Dyn
amic
mo
du
lus
Alginate Concentration (%)
4B20 Laboratory- Crosslinking of Alginate for Biomaterial Applications 5
V. CONCLUSIONS
The ionic and covalent cross-linking of alginate provides
extensive biomaterial applications. Ranging from areas such
as wound healing (through beads) to drug delivery (shape
memory foam, microspheres) and tissue engineering (stem cell
carriers). The author believes that further research into the
cross-linking of alginate, and particularly into its shape
memory properties, would positively impact the biomedical
industry.
VI. REFERENCES
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p. 153-155 [2] Tonnesen H.H. and Karlsen J. Alginate in Drug Delivery Systems, Drug
Development and Industrial Pharmacy, 2002, 28(6), p. 621-630
[3] Li, Zhensheng, et al. Chitosan–alginate hybrid scaffolds for bone tissue engineering, Biomaterials, 2005, 26(18): p. 3919-3928.
[4] Buckley C.T ., Hydrogel Systems, 4B20 Biomaterials lecture notes, 2014, p. 16
[5] Ashton R.S. Banerjee A, Punyani S, Schaffer D.V. Kane R.S. Scaffolds based on degradable alginate hydrogels and poly(lactide-co-glycolide) microspheres for stem cell culture, Biomaterials, 2007, 28(36): p. 5518-5525
[6] Kondo A. and Fukuda H., Preparation of thermo-sensitive magnetic hydrogel microspheres and application to enzyme immobilization, Journal of Fermentation and Bioengineering, 1997, 84(4): p. 337-341
[7] Mathiowitz E. et al. Biologically erodible microspheres as potential oral
drug delivery systems, Nature, 1997, 386: p. 410-414 [8] Buckley C.T ., Alginate Hydrogels, 4B20 Biomaterials lecture notes,
2014, p. 14-15 [9] Ouwerx C, Velings N, and Mestdagh M.M. Physico-chemical properties
and rheology of alginate gel beads formed with various divalent cations, Polymer Gels and Networks, 1998, 6(5): p. 393-408
[10] Klokk T .I. and Melvik J.E., Controlling the size of alginate gel beads by
use of a high electrostatic potential, Microencapsulation, 2002, 19(4): p. 415-424
[11] Vandenberg G.W. and De La Noue J, Evaluation of protein release from chitosan-alginate microcapsules produced using external or internal
gelation [12] LeRoux M.A., Guilak F, Setton L.A., Compressive and shear properties
of alginate gel: Effects of sodium ions and alginate concentrations, 1999, in press
[13] Buckley C.T ., Alginate Hydrogels, 4B20 Biomaterials lecture notes, 2014, p. 22-29
[14] Sokolowski W, Metcalfe A, Hayashi S, Yahia L.H. and Raymond J, Medical Applications of Shape Memory Polymers, Biomedical Materials,
2007, 2(1): p. 23