J. Biomedical Science and Engineering, 2011, 4, 383-390
doi:10.4236/jbise.2011.45048 Published Online May 2011 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online May 2011 in SciRes. http://www.scirp.org/journal/JBiSE
In vitro degradation behavior of chitosan based hybrid
microparticles
Ambalangodage C. Jayasuriya, Kristalyn J. Mauch
Department of Orthopaedics, College of Medicine, University of Toledo Health Science Campus, Toledo, USA.
Received 9 February 2011; revised 9 March 2011; accepted 21 March 2011.
ABSTRACT
The degradation properties of the MPs is important
to the long-term benefits of the use of the chitosan
(CS) based hybrid MPs in bone tissue-engineering,
because the degradation kinetics could affect a mul-
titude of processes within the cell, such as cell growth,
tissue regeneration, and host response. The aim of
this study was to investigate the degradation of solid,
hybrid CS microparticles (MPs), CS-10% calcium
phosphate (CaHPO4, w/w), and CS-10% calcium
carbonate (CaCO3, w/w) MPs in phosphate buffered
solution (PBS) over a 30-week period. The hybrid
MPs were synthesized by emulsification technique,
cross-linked with 64% sodium tripolyphosphate (TPP),
purified and air dried overnight. Each sample had 30
mg of MPs was placed in a glass vial with 9 ml of PBS
added and then the vial was closed to prevent evapo-
ration. Every week 4 ml of the incubated solution was
removed for sample measurement and all samples
were replaced with an equivalent amount of fresh
medium. The samples were maintained at 37˚C under
continuous shaking. The hybrid MPs were measured
for pH and calcium release, every week in triplicate.
At 0, 5, 10, 15, 20, 25, and 30 weeks, surface and bulk
morphology were analyzed with a scanning electron
microscope (SEM). The degradation data suggested
that the hybrid MPs were stable at least up to 25
week and maintain the physiologically relevant pH.
Therefore, we can use these hybrid MPs to apply in
the bone tissue engineering applications since they do
not degrade within a short period.
Keywords: Degradation; In Vitr o; Microparticles; pH;
Chitosan; Phosphate Buffered Saline
1. INTRODUCTION
Scaffolds are typically used in bone tissue engineering,
acting as a delivery system for the cells, genetic material,
and growth factors to the site of interest or defect [1].
Bone tissue engineering involves combining bone-
forming cells with a suitable scaffold. The scaffold is
needed to function as a material for support, and a sub-
strate for cell attachment and bone deposition [2,3]. The
ultimate aim of bone tissue engineering is to help create
a healing response in the bony area designated so that
newly formed tissue would integrate with the surround-
ing skeleton to be useful and resilient. Ideally, the engi-
neered scaffold will slowly degrade in a controllable
manner. Controllable in the sense that degradation would
match extracellular matrix formation, transfer structural
and functional roles progressively to the newly formed
bone while maintaining mechanical strength until tissue
regeneration is almost completed, and finally be reab-
sorbed and metabolized by the body. The degradability
of the biomaterial plays an important role in the
long-term function of the engineered scaffold because it
can affect many cellular process, including cell growth,
tissue regeneration, and host response [1,4,5]. To find a
suitable candidate that encompasses these aims, a variety
of biomaterials have been researched for bone tissue
engineering purposes, including biomaterials that can be
synthetic or natural [3,5].
As natural components of living structures, naturally
derived polymers are of particular interest as a scaffold
material for bone tissue engineering applications due to
the biological and chemical similarities that they have to
natural tissues. The supposition being that natural mate-
rials would have better acceptance by the human system
because of its natural origins and having a closer relation
to the body’s original components. Chitin and its deriva-
tive chitosan (CS) are such natural biomaterials that can
meet a variety of needs in the biomedical fields because
their biological, physical, and chemical properties can be
controlled and engineered under even mild processing
conditions [4,6]. Mild processing conditions can avoid
growth factor inactivation, allowing growth factors and
other biomolecules to be incorporated into various com-
posite forms for different applications [5]. Another fa-
vorable property of the derivative CS is that it can be

A. C. Jayasuriya et al. / J. Biomedical Science and Engineering 4 (2011) 383-390 384
molded into different forms and shapes; for example,
powders, pastes, films, fibers, sponges, scaffolds, mi-
croparticles (MPs), and more have been made for vari-
ous uses [7,8]. All these factors therefore lend credence
to why chitin and CS are being increasingly studied for
diverse applications as biomaterials in biomedical and
pharmaceutical research, ranging from wound dressings
to drug delivery carriers, as well as being used for cell
encapsulation, or cartilage and bone tissue engineering
[6,9].
CS is a linear copolymer that is formed by the random
distribution of D-glucosamine and N-acetyl-D-gluco-
samine residues that are linked by
 (1-4) glycosidic
bonds. CS is not native to animal sources, however it is
easily obtained from crustacean’s exoskeletons, such as
shrimp and crab, by alkaline deacetylation of chitin
[6,8,10,11]. Removing acetyl groups from the molecular
chain of chitin leaves an amino group on the chain spe-
cifically at the C-2 carbon of the glucopyranose ring in
the deacetylation process of chitin to CS. However, nei-
ther chitin nor CS respectively exist 100% acetylated or
100% deacetylated, but both exist as a copolymer.
Therefore, the difference between chitin and CS is the
acetyl content of the copolymer. The degree of acetyla-
tion refers to the number N-acetyl-D-glucosamine units
that are present and when greater than 50%, the copoly-
mer is termed chitin. When the D-glucosamine (amino
groups) are predominant (>50%) the copolymer is
termed CS. The number of amino groups present refers
to the degree of deacetylation [6]. Chitin’s use is limited
compared to CS because chitin is chemically inert and is
insoluble in water and acid. CS is insoluble in neutral
and basic pH environments (pH > 7), and soluble in
acidic environments (pH < 6) when the amino groups
become protonated to facilitate solubility [7,12,13].
Besides CS being an easily obtained derivative of a
natural copolymer that is moldable with reactive func-
tionalities, the investigation into CS being used as a
scaffold material in bone tissue engineering is largely
due to a number of beneficial biological properties in-
cluding being nontoxic, biocompatible, and biodegrad-
able [3,10,11,14]. CS has been shown to have intrinsic
antimicrobial actions on bacteria and fungi, to be
haemostatic, antitumoral, and anticholestermic. Having
an affinity to proteins, promoting cell adhesion and mi-
gration, and enhancing wound healing list a few more
favorably reported properties of CS [4,11]. CS also has
been shown to promote growth and mineral rich matrix
deposition by osteoblasts in culture. Implants that are CS
based have generally shown a minimal foreign body
reaction with little or no fibrous encapsulation, which is
a helpful property that could be useful to the osteointe-
gration important in bone tissue engineering that can
lend significant rigidity to bone-implant systems [4,12,
13,15].
In this study, the aim was to study the degradation
behavior of hybrid MPs at physiological conditions.
Since the inorganic portion of bone is composed mainly
of calcium and phosphate [3], hybrid MPs were also
made containing CaHPO4 or CaCO3 individually to
compare and see if hybrid MPs could offer any specific
advantages. Regarding this, we fabricated three different
types of hybrid MPs including CS, CS-10% calcium
phosphate (CaHPO4), and CS-10% calcium carbonate
(CaCO3) and placed in the glass vials containing phos-
phate buffered saline (PBS) medium with pH = 7.4.
These vials containing MPs were incubated at 37˚C with
dynamic environment (50 rpm), and refreshed medium
frequently during the study. Our degradation behavior of
different types of hybrid MPs was done by measuring
pH and calcium release, and analyzing both surface and
bulk morphology using scanning electron microscopy
(SEM) over an extended time period of 30 weeks.
2. MATERIALS AND METHODS
2.1. Materials
CS from crab shells, practical grade and > 85% deacety-
lated, was purchased from Sigma-Aldrich. Other chemi-
cals bought from Sigma-Aldrich (St. Louis, MO, USA)
include: cottonseed oil, CaHPO4, CaCO3, sodium tri-
polyphosphate (TPP), Span 85, hexane, and acetic acid.
Acetone was supplied by Fisher Science (Hanover, IL,
USA). GIBCO PBS 7.4 (1X) liquid from Invitrogen
(Grand Island, NY, USA) was used throughout the ex-
periment. QuanticChrom calcium Assay kit was used
from BioAssay Systems (Hayward, CA, USA). All sol-
vents and chemicals used, unless specified were of ana-
lytical grade.
2.2. Hybrid MP Preparation
CS MPs (1.5%, w/v) were prepared by emulsification
technique using our scale-up method [16,17]. CS solu-
tion was prepared by measuring 750 mg of CS that was
diluted by adding 50 mL of 1% (v/v) acetic acid at room
temperature. Using a stir plate, the mixture was allowed
to reach homogeneity and then it was filtered through a
nylon mesh 50 micron cloth to remove any remaining
insoluble particulates. The CS solution (25 ml) was
quickly mixed with an equal amount (25 ml) of acetone.
This CS-acetone mixture (36 ml) was emulsified by
adding the mixture drop wise using 20 ml syringes
(Becton Dickinson [B-D], Franklin Lakes, NJ) and 20
gauge 1’1/2” needles (B-D) into mechanically stirred
cottonseed oil (600 ml) that included surfactant Span 85
(4 ml) at 35˚C - 40˚C and 850 rpm (Corning Laboratory
Stirring/Hotplate model, USA). The system continued
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A. C. Jayasuriya et al. / J. Biomedical Science and Engineering 4 (2011) 383-390 385
stirring for 14 h to allow evaporation of the non-oil sol-
vent. Then sodium tripolyphosphate (64%, TPP) was
added to allow cross-linking of the MPs for 4 h (Figure
1). CS MPs were washed with an equal amount of hex-
ane (600 ml), isolated by vacuum filtration (11 cm me-
dium porosity filter paper, Fisherbrand, Pittsburgh, PA,
USA) with a Buchner funnel, then air dried overnight.
We also fabricated MPs containing CaHPO4 or CaCO3
(10%, w/w solution of polymer) by adding them into the
CS solution and using above described method. All pre-
pared MPs were hydrated and neutralized in 6200 mg/L
NaHCO3 for 10 min. The solution was filtered off and
the MPs were then rinsed profusely with distilled water
and allowed to air-dry overnight.
2.3. Experiment Design of in Vitr o MP
Degradation
The fabricated and neutralized hybrid MPs (30 mg) were
added to 11 ml glass vial shells (Fisherbrand). MPs sam-
ples were incubated with 9 ml of Phosphate Buffered
Saline (PBS) at 37˚C (Fisher Scientific Isotemp Incuba-
tor) under continuous shaking (Barnstead Multi-Purpose
Rotator) at 50 rpm for predetermined time points up to
30 weeks. The vials were closed with a plastic plug de-
signed for the vials, to prevent evaporation of the PBS
over the long time period of the experiment. In order to
mimic in vivo physiological environment, every week 4
ml of incubated PBS solution was removed from each
sample and replaced with an equal volume of fresh PBS
medium.
2.4. Determination of pH
CS and hybrid MPs were studied in triplicate samples
for pH determination. Each week the 4 mL of PBS in-
cubated solution withdrawn from the MPs was removed
from their vial and placed in clean vial shells to measure
the pH and help keep the sample from contamination.
The pH was determined by placing a clean and cali-
brated pH meter (Mettler Toledo SevenEasy) probe into
the solution then recording the stabilized value.
2.5. De t e r m i n a tio n of C a l c i u m I o n ( C a 2+) Release
From the removed PBS medium used in pH determina-
tion, two 0.5 ml aliquots of the incubated medium were
stored at 4˚C for assay analysis. The stored aliquots and
assay kits were removed from the freezer and allowed to
equilibrate to room temperature before use. The calcium
assay requires preparation of a working reagent by com-
bining equal volumes of the Reagent A and Reagent B
provided. Standards, blanks, and samples were run in
triplicate on clear bottom 96-well plates by adding 5 l
of sample and then 200 l of working reagent to each
well with tapping lightly to mix. Samples were allowed
Figure 1. Ionic cross-linking interaction of amide groups in
chitosan with phosphate groups in tripolyphosphate.
to incubate 3 min at room temperature and the optical
density (OD) was read at 612 nm on a spectrometer
(Spectra Max Plus 384, Molecular Devices; Sunnyvale,
CA). Calcium concentrations of the samples were calcu-
lated with the following expression using the average
OD of all triplicates of the samples:
Calcium Concentration = SAMPLE BLANK
OD OD
Slope
 (mg/dL)
2.6. Morphology of MPs-SEM
At the predetermined time points of 0, 5, 10, 15, 20, 25,
and 30 weeks, samples were removed for characteriza-
tion by SEM. Size and morphological characteristics,
including shape and surface roughness of the fabricated
hybrid MPs were studied with a Hitachi S-4800 High
Resolution SEM at an accelerating voltage of 10 kV.
Samples for analysis with the SEM were prepared as
follows: small amounts of dried MPs were affixed to a
stage on double sided tape then the dry samples were
gold sputtered prior to analysis with the SEM. Micro-
photographs were taken at different magnifications for
each sample for analysis.
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2.7. Statistical Analysis
Statistical analysis was performed on pH and calcium
release with a two-way ANOVA without replication
through the Analysis ToolPak for data analysis in Excel.
The differences were considered significant at the level
of p < 0.05.
3. RESULTS
3.1. Study of pH
We recorded the averaged pH changes (n = 3) for the
different types of MPs when they immersed in the PBS
medium over the 30 weeks (Figure 2). In the figure it is
clear that the MPs are causing changes in the pH of the
PBS medium. Each MP type has a specific pH range that
varies over time, but stays within the range that is com-
patible for living tissue [18]. CS MPs kept the highest
pH range among all MP types, while CS-10%CaHPO4
MPs had the lowest pH range (p < 0.05) for most of the
study. While CS MPs maintained the pH range in 7.4 -
7.2, CS-10%CaCO3 MPs maintained the pH in 7.2 - 7.0.
CS-10%CaHPO4 MPs showed the lowest pH range, 6.8 -
7.0 among the three MP groups.
3.2. Calcium Ion (Ca2+) Release
We fabricated MPs that integrated CaHPO4 or CaCO3
into their own respective structures as they formed. The
calcium ion release in mg/dl is reported in Figure 3 over
the 25 weeks for each type of hybrid MPs (n = 3). There
was no calcium release during the 4 weeks period from
CS MPs. CS-10%CaCO3 MPs have the highest variation
of calcium release during the initial 6 weeks compared
to all the groups (p < 0.05). Calcium release from
CS-10%CaCO3 MPs was decreased at 18 week and then
again started to increase (p < 0.05). Other two types of
MPs was also followed the same pattern after 18 week.
Calcium release in the surface of the CS-10%CaCO3
MPs is seems to be faster than compared to that of
CS-10%CaHPO4 MPs.
3.3. Morphology Change—SEM
The size, shape, and surface roughness of MPs can in-
fluence cell attachment, proliferation, and differentiation
[2]. SEM microphotographs were used to determine
these characteristics of hybrid MPs. Almost prepared
MPs appeared to be spherical and distinctively separate
at Week 0, which means just after placing the MPs in the
vials containing PBS for 10 - 15 min but not yet started
to incubate them (Figures 4-6). All MPs types were
measured had their smallest diameter approximately 20
m. The most CS MPs were in the size range between
20 - 75 m. However, there were a few MPs were over
the upper limit of the size range. The CS-10%CaCO3
and CS-10%CaHPO4 MPs were slightly larger compare
to the CS MPs and the most MPs were in the size of 20 -
90 m, and 20 - 100 m, respectively. Similar to CS
MPs there were a few large 10%CaCO3 and CS-10%
CaHPO4 MPs in the out of above size range.
In Figure 4, microphotographs show the changes in
surface morphology of CS MPs with low (50X) and high
magnification (700X). Week 0 had smooth, spherical
morphology with slight ridges elevations. At Week 5 we
noticed a change to the outer surface morphology of
most CS MPs. Larger ridges and wrinkling were ob-
served. The wrinkles seem to become more predominant
as the time points passed. At Week 25, we were inter-
ested that the microphotographs revealed that the some
MPs were peeling but the shape was not affected. At
week 30, small pieces were broken from the some CS
MPs. However, we did not observe the complete bulk
degradation of MPs suggesting that bulk degradation
initiate at this time period.
Figure 2. The pH data for hybrid MPs as a function of time up
to 30 week. The line with solid spheres shows the pH of the
replacement PBS medium used.
Figure 3. Calcium release data for each type of MPs over the
25-weeks.
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A. C. Jayasuriya et al. / J. Biomedical Science and Engineering 4 (2011) 383-390 387
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 4. SEM microphotographs of CS MPs from
weeks (a) 0; (b) 5; (c) 10; (d) 15; (e) 20; (f) 25; and (g)
30. 1 and 2 denote low (50X) and high (700X) magni-
fication, respectively. Arrows indicate the degradation
started areas.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 5. SEM microphotographs of CS-10%CaHPO4
MPs from weeks (a) 0; (b) 5; (c) 10; (d) 15; (e) 20; f) 25;
and (g) 30. 1 and 2 denote low (50X) and high (700X)
magnification, respectively. Arrows indicate the degra-
dation started areas.
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A. C. Jayasuriya et al. / J. Biomedical Science and Engineering 4 (2011) 383-390 388
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 6. SEM microphotographs of CS-10%CaCO3
MPs from weeks (a) 0; (b) 5; (c) 10; (d) 15; (e) 20; f) 25;
and (g) 30. 1 and 2 denote low (50X) and high (700X)
magnification, respectively. Arrows indicate the degra-
dation started areas.
Microphotographs of CS-10%CaHPO4 MPs (Figure 5)
revealed spherical particles that tended to stay separate,
but have an increasingly rougher surface with the pas-
sage of time. However, the surface is considerably
smoother than CS MPs. Small holes can also be observed
in the CS-10%CaHPO4 MPs especially after Week 20.
At week 25, the particle size and shape were not affected
but at week 30, the pieces of the most particles were
observed suggesting the initiation of bulk degradation.
Figure 6 shows the CS-10%CaCO3 MPs microphoto-
graphs (50X and 700X) that reveal a distinctive flaky
Week 0 appearance. Beginning Week 5 the noticeable
flaky appearance was mostly gone, exhibiting a smoother
surface that was more comparable to CS MPs from week
0. The flakiness appearance went away and the surface
did get mildly rougher over time. The initial flakiness
may be attributed to the leaching of CaCO3, TPP, or both.
At 25 and 30 weeks, breaking of parts and appearing of
holes can be visible in some CS-10%CaCO3 MPs.
4. DISCUSSION
We fabricated the ionically cross-linked hybrid MPs
(Figure 1) in this study using our optimized scale up
method as described previously [16]. We also character-
ized these hybrid MPs in terms of chemical composition
and ionically cross-linked structure, physical structure
and morphology using different analytical techniques
[17]. The hybrid MPs are spherical in shape with smooth
outer surface without porous structure. The most MPs
fabricated were in the range of 20 - 100 m.
Instead of static conditions of the degradation, we
performed degradation studies similar to in vivo condi-
tions, for example, kept the vials containing MPs in PBS
at pH 7.4 in a shaking table (50 rpm) inside the incubator
at 37˚C. The incubated medium in the vials was also
refreshed frequently. Before using these hybrid MPs to
study drug release profiles or in vivo studies, it is neces-
sary to know their biodegradation behavior in the
physiological environment.
The pH data of hybrid MPs during 30 week period
was in the range of 6.8 - 7.4. The CS MPs has shown the
highest pH values (7.2 - 7.4) during the 30 week period
suggesting that CS-10%CaHPO4 MPs involves slightly
lower values (6.8 - 7.0) of pH compared to other two
types of MPs. These lower pH values of 10%CaHPO4
MPs may be related to the release of H+ ions from
CaHPO4 in 10%CaHPO4 MPs. Hydrogen ion concentra-
tions outside of the tenfold pH range of 7.8 to 6.8 are not
compatible with living tissue if maintained [18]. The pH
of these hybrid MPs over 30 week period fulfilled the
above requirement. However, we anticipate that we can
improve the higher pH values if we use less acetic acid
when we fabricate the MPs. In addition we must use the
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A. C. Jayasuriya et al. / J. Biomedical Science and Engineering 4 (2011) 383-390 389
lyophilizer to get rid of any remaining residual acetyl
group in the MPs.
We examined the calcium release for all types of MPs
during the 25 week period (Figure 3). The calcium re-
lease from MPs was less than 2 mg/dl at a time during
the 25 week period. This released calcium amount seems
to be less than the calcium content in the human serum
which is 8.5 - 10.5 mg/dl [19]. Our MPs contain only
10% (w/w) of CaHPO4 or CaCO3 relative to CS amount.
We can anticipate that higher release of calcium at a time
if we incorporate the higher amount of calcium contain-
ing compounds into the hybrid MPs. The initial calcium
release seems to be attributing to the diffusion and later
release after 18 week may be due to the more degrada-
tion of the MPs. The CS MPs may not possess calcium
since we did not incorporate any materials containing
calcium. However, our data has shown the calcium re-
lease over time from CS MPs. We would like to note that
Chestnutt et al. [20] also reported the interesting release
of calcium in pure CS scaffolds. A fact that was attrib-
uted to the use of CS from crab shells containing resid-
ual mineral content.
SEM data (Figures 4-6) has clearly shown that these
MPs did not change its shape or size until 25 week. At or
after 25 week all types of MPs seem to show the initia-
tion of bulk degradation. Generally, the degradation of
CS is related to the molecular weight and deacetylation.
These MPs were strong not only having the higher
deacetylation (>85%) but these MPs were ionically
cross-linked with TPP. This interaction leads to the fab-
rication of physically strong particles according to our
optimization and scale-up procedures [16].
It has been demonstrated that CS can be degraded en-
zymatically by chitinases, chitosanases, and lysozyme.
Past research has shown CS to be degraded mainly by
lysozyme in human serum [21,22]. Lysozyme appears to
target the acetylated units on CS, leaving CS oligosac-
charides products of different lengths that can be incor-
porated into glycosaminoglycans and glycoproteins
pathways or excreted [13,23]. Many investigations have
published that the factor in controlling the rate of degra-
dation has been shown to be inversely associated with
CS’s degree of deacetylation. Highly acetylated CS de-
grades rapidly, and highly deacetylated (>80%) CS has
low degradation rates and may remain several months in
vivo [5,6,10,12,13,22].
There is an increased demand towards surgical inter-
ventions being minimally invasive. Preferable scaffold
constructs in bone tissue engineering for such surgeries
would be moldable and/or injectable to the defect site.
Some studies have shown the possibility of such osteo-
genic constructs with pre-cultured ceramic MPs. How-
ever, low biodegradability and brittleness can restrict the
use of ceramics [2,4]. A goal of our studies is to help
characterize and design CS based hybrid MPs that could
become an injectable biomaterial suitable for a scaf-
fold-like role in bone tissue engineering. We believe it to
be important to understand the process that is happening
to the MPs without enzyme for background results that
can be used comparatively with what happens with en-
zymatic degradation in future studies.
5. CONCLUSIONS
In this study using physiological conditions we have
shown that morphological changes occur on the surface
followed by initiation of bulk degradation of 85%
deacetylated CS based hybrid MPs without enzyme hy-
drolysis over 30 week period. The size and shape of the
hybrid MPs were not affected until week 30. The broken
pieces were observed at Week 30 from the all types of
hybrid MPs. Our results show that each MP type main-
tain the physiologically relevant pH range during the 30
week period. We conclude that investigation of using CS
based hybrid MPs for a bone tissue engineering purpose
is encouraged by our biodegradation results, and the
feasibility of using these MPs for a long-term scaf-
fold-like role is possible.
6. ACKNOWLEDGEMENTS
We would like to thank National Science Foundation (NSF) grant
number 0652024 and National Institute of Health (NIH) grant number
DE019508 for providing financial support to accomplish this work.
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