Vietnam National Universities – HCMC

International University
School of Biomedical Engineering

Course of Biomaterials
Semester 1 2022-2023

LAB #3
NATURAL POLYMER (CHITOSAN)

I. Outline
 Overview of chitosan material.
 Overview of hydrogel formation technique.
 Overview of chitosan oligosaccharide.
 Experimental protocols.
 Report highlights.

II. Chitosan material

 Chitosan is modified from chitin by NaOH treatment. Chitin is the second most
abundant polysaccharide in nature (after cellulose) and derived mainly from
exoskeletons of crustaceans (such as crabs and shrimps).

Fig 1. Chemical structure of chitin

Fig 2. Chemical structure of chitosan

 Vietnam National Universities – HCMC
International University
School of Biomedical Engineering

III. Hydrogel formation technique

 Hydrogel is a well-known structure for various biomedical applications, such as scaffold
for tissue engineering and 3D bioprinting, drug and stem cell delivery system, etc.
Hydrogel is formed by crosslinking hydrophilic polymer chains together by non-covalent
bond (physical crosslinking) or covalent bond (chemical crosslinking), which results in an
elastic, gel-like and mostly adhesive structure with water inside. It greatly resembles the
extracellular matrix nature of living tissue and may respond to the environment (such as
changing pH, changing temperature, etc.).
 The fundamental principle of hydrogel formation includes (1) preparing a polymeric
solution in water and (2) triggering crosslinking reaction in the solution, by physical means
(such as pH alteration, temperature alteration and ionic interaction) or chemical means
(use of crosslinker, use of reactive polymer components, light exposure at a specific
wavelength, etc.)
 Safety related to crosslinker hazard should be noted.

Fig 2. Illustration of a hydrogel

IV. Chitosan oligosaccharide (COS)

 Chitosan has been proved to exert antimicrobial property by different underlying
mechanisms, one of which is associated with the existence of positive charges (-NH3+) on
the polymer chains that helps disrupt bacteria membrane of both strains.
 However, chitosan is not soluble in water at neutral pH, which usually requires a
modification step to improve its solubility. One of the techniques involves cleaving the
long polymer chains into shorter ones, creating chitosan oligosaccharide (COS). This can
be achieved by utilizing an oxidant (such as hydrogen peroxide) and microwave-assisted
irradiation.

V. Experimental protocols
1. Hydrogel formation
 Vietnam National Universities – HCMC
International University
School of Biomedical Engineering

1.1. Physical crosslinking – pH alteration

Step 1: Prepare chitosan solution at 2% (w/v) concentration in distilled water with acetic acid
(2% v/v) in a glass vial.
Step 2: Prepare sodium hydroxide (NaOH) aqueous solution at 2.5 M and hydrochloric acid
(HCl) at 2.5 M.
Step 3: Add drops of NaOH solution into chitosan solution while stirring until it forms hydrogel.
Record the required volume of NaOH solution. Add drops of hydrochloric acid while stirring
to see if the hydrogel can be converted back to solution state. Record the required volume of
HCl in case that the conversion takes place.
1.2. Chemical crosslinking – use of crosslinker
Step 1: Prepare chitosan solution at 2% (w/v) concentration in distilled water with acetic acid
(2% v/v) and add 1 ml of it into the well of 24-well plate for three times.
Step 2: Prepare glutaraldehyde (GTA) solution at different concentrations of 1, 5 and 10%
(w/v).
Step 3: Add 0.2 ml of each GTA solutions into the corresponding well and manually mix to
see if a hydrogel structure can be formed or not.
2. COS synthesis
Step 1: Prepare 50 ml of H2O2 6% (w/w) from stock solution (30% w/w).
Step 2: Immerse chitosan powder into the H2O2 solution above at 6% (w/v). Stir for 15 minutes
using magnetic stirrer.
Step 3: Seal the beaker and create some holes on the seal. Irradiate the mixture using a
microwave until complete dissolution. Take the mixture outside every time the solution boils
to reduce the temperature and solvent evaporation. Cool the mixture to room temperature
afterwards.
Step 4: Add absolute ethanol to obtained solution at a volume ratio of 3:1. Centrifuge the
solution at 12,000 rpm in 20 minutes at 4oC. Collect the precipitate into a petri-dish, freeze,
lyophilize and store the dried powder at 2-8oC.

VI. Report highlights

 Introduction section should include brief information of:
+ Background of natural polymer (compared to synthetic polymer) and its use as
biomaterial.
+ Background of chitosan material.
+ Background of hydrogel (advantages/ disadvantages, crosslinking techniques, etc.)
+ Background of COS material and COS preparation methods.
 Vietnam National Universities – HCMC
International University
School of Biomedical Engineering

 Questions (for Result and Discussion section)

1. Do we need to consider the conditions of the crosslinking step to form hydrogel (such
as pH, temperature, use of toxic crosslinker, etc.) if the hydrogel is targeted to be
applied in tissue engineering field and to load cells/ stem cells?
2. What is the suitable molecular weight of chitosan to be considered as COS?