4B20 Lab-Chirag Chadha-11334006

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:

[email protected]).

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


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


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.


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.


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.



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


=

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


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.


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.


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




Equilibrium modulus over 0.5%, 1%, and 2% concentrations

Dynamic modulus over 0.5%, 1%, and 2% concentrations



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


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.


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]


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]



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.



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 (%)


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

[1] Barnett S.E. and Varley S.J. The effects of calcium alginate on wound healing, Annals of the Royal College of Surgeons of England, 1987, 69:

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

4B20 Lab-Chirag Chadha-11334006

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