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Acta Biomaterialia 5 (2009) 328–337

www.elsevier.com/locate/actabiomat

Gelatin/chitosan/hyaluronan scaffold integrated

with PLGA microspheres for cartilage tissue engineering
Huaping Tan, Jindan Wu, Lihong Lao, Changyou Gao *
Key Laboratory of Macromolecular Synthesis and Functionalization, Ministry of Education,
and Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China

Received 16 January 2008; received in revised form 9 July 2008; accepted 15 July 2008
Available online 6 August 2008

Abstract

Poly(lactide-co-glycotide) (PLGA) microspheres integrated into gelatin/chitosan/hyaluronan scaffolds were fabricated by freeze-dry-
ing and crosslinking with 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. The effects of the microspheres on porosity, density, com-
pressive modulus, phosphate-buffered saline uptake ratio and weight loss of the scaffolds were evaluated. Generally, a scaffold with a
higher PLGA content had a lower porosity and weight loss, and a medium uptake ratio, but a larger apparent density and compressive
modulus. When the PLGA content was lower than 50%, the PLGA-integrated scaffolds had a similar pore size (200 lm) as that of the
control, and as much as 90% of their porosity could be preserved. In vitro chondrocyte culture in the 50% PLGA-integrated scaffold
demonstrated that the cells could proliferate and secrete extracellular matrix at the same level as in the control gelatin/chitosan/hyalu-
ronan scaffold.
Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Microspheres; PLGA; Scaffolds; Compressive properties; Chondrocytes

1. Introduction good mechanical properties, biodegradability and process-

Cartilage damage occurs frequently owing to sports and
progressive ageing, and cannot be spontaneously repaired.
In recent years, cartilage regeneration has been successfully
achieved by tissue engineering and regenerative medicine,
in which apart from the seed cells and tissue construction,
the temporary scaffold plays an important role in guiding
the cell adhesion and proliferation, and maintaining the
normal phenotype of the chondrocytes [1–4]. Along with
the secretion of extracellular matrix, mostly collagen type
II and glycosaminoglycans (GAGs), the scaffold is progres-
sively degraded and the cartilage is ultimately regenerated.
Many kinds of synthetic and natural polymer materials
have been used as scaffolds for tissue regeneration. Syn-
thetic polymers such as poly(lactic acid) (PLA), poly(gly-
colic acid) (PGA), and their copolymers (PLGA) have
*
Corresponding author. Tel.: +86 571 87951108; fax: +86 571 87951948.
E-mail address: cygao@mail.hz.zj.cn (C. Gao).
ibility, but have poor cell–matrix interaction [5–10]. By
contrast, natural biomaterials such as collagen, gelatin,
chitosan, chondroitin and hyaluronic acid can maintain a
differentiated cell phenotype and allow rapid cell expansion
[11–14]. However, they have poorer mechanical stability
and a faster rate of biodegradation [12–13].
A scaffold with a similar physical structure and chemical
composition as that of the cartilage matrix is highly desirable
for maintaining the proliferation and differentiation of chon-
drocytes, so that the regenerated cartilage can have normal
biological functions and mechanical strength. A simple but
effective way to obtain such a scaffold is to integrate naturally
originating biomacromolecules and synthetic polymers,
which take the role of natural extracellular matrix (ECM)
and endow the scaffold with stronger strength. It is known
that in hyaline cartilage type II collagen (15–20%), chondroi-
tin sulfate (5–10%) and hyaluronan (0.05–0.25%) are the
main ECM components, which further assemble into a
supramolecular structure that has good mechanical strength

1742-7061/$ - see front matter Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.actbio.2008.07.030
 H. Tan et al. / Acta Biomaterialia 5 (2009) 328–337
[14]. So far it is hard to mimic completely the supramolecular
structure, but it is not difficult to mimic the chemical compo-
sition by using naturally originating biomacromolecules
such as gelatin, chitosan and hyaluronan [15].
Gelatin is a partial derivative of collagen. It is nonimmu-
nogenic compared to its precursor and presumably retains
informational signals such as the RGD sequence, and thus
can promote cell adhesion, differentiation and proliferation
[11,12]. Chitosan, a partially deacetylated derivative of chi-
tin, contains glucosamine and N-acetylglucosamine, and is
structurally similar to GAG. Hyaluronan is a water-soluble
polysaccharide that is widely distributed throughout the
ECM of all connective tissues in mammals. It plays an
essential role in the maintenance of the normal ECM and
is a chondroinductive agent that modulates the pericellular
matrix during embryonic cartilage development. Results
show that this ternary complex scaffold can effectively
maintain the chondrocyte phenotype and support chondro-
genesis in vitro [15]. However, as with most scaffolds
obtained from natural materials, this scaffold has poor
mechanical properties and quickly loses its shape and size.
In this work, we attempt to integrate synthetic polymer
microspheres into the ternary complex to obtain a compos-
ite scaffold. For this purpose, PLGA was chosen as the
material to formulate the microspheres because of its better
biocompatibility, degradability and processibility.
Microspheres are attracting increasing interest because
of their diverse applications in the field of drug [16–19]
and growth factor [20–23] delivery for tissue engineering.
It has been reported that implantation of microspheres
containing growth factors resulted in improved cell pheno-
type and chondrogenesis [24–28]. Although the bioactivity
following growth factor implantation is impressive, the
insufficient support for the newly formed ECM could fail
to reconstitute a well-remodeled cartilage surface zone
and might tend to deteriorate with time. We shall also show
in this work that microsphere integration is a convenient
way to improve the compressive strength of the natural
polymer scaffolds, without affecting the biocompatibility.
2. Materials and methods
2.1. Materials
Gelatin and chitosan (percentage of deacetylation 85%,
Mg = 6.2  105) were obtained from Shanghai Chemical
1)
a b
PLGA microsphere Gelatin/chitosan/hyaluronan matrix
Scheme 1. Schematic illustration to show the preparation of PLGA microsphere-integrated scaffold. (a) Gelatin/chitosan/hyaluronan solution; (b) the
solution was integrated with PLGA microspheres and cross-linked with EDAC; (c) the porous scaffold was obtained after lyophilization.
329
Industries Co. Ltd. and Qingdao Haidebei Bioengineering
Co. Ltd. (China), respectively. Sodium hyaluronate, 1-
ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC),
collagenase type II, fluorescein diacetate (FDA), 1,9-dim-
ethylmethylene blue and chondroitin sulfate were pur-
chased from Sigma. PLGA (85/15 lactide/glycolide ratio,
Mn = 108 kDa, Mw = 203 kDa) was purchased from China
Textile Academy. Poly(vinyl alcohol) 124 (PVA 124, Mw
85–124 kDa, 98–99% hydrolyzed) was supplied by Shang-
hai Medicine and Chemical Company, China. All other
chemicals and bioreagents were used as received without
further purification.
2.2. Preparation of PLGA microspheres
The PLGA microspheres were fabricated using an emul-
sion solvent evaporation technique. Briefly, a PLGA solu-
tion (250 mg in 5 ml methylene chloride) was added to
50 ml of a stirred 0.5% (w/v) PVA aqueous solution to
form a multiple emulsion. The multiple emulsion was stir-
red for 24 h (1000 rpm) at room temperature to evaporate
the organic solvent. The PLGA microspheres were col-
lected by centrifugation (2000 rpm for 5 min), washed five
times with triple-distilled water to remove PVA and then
lyophilized for 24 h. The dried microspheres were stored
at 4 °C prior to usage.
2.3. Scaffold preparation
The PLGA particle-integrated scaffolds were prepared
by a freeze-drying technique (Scheme 1). In brief, 0.5 g gel-
atin powder and 5 mg sodium hyaluronate were dissolved
in 8 ml triple-distilled water. After agitation for 2 h at room
temperature, 2 ml 5% (w/v) chitosan/0.5 M acetic acid
solution was added. After continuous agitation for 1 h,
the PLGA microspheres were added into this gelatin/chito-
san/hyaluronan ternary solution. The final weight ratio of
the microspheres was controlled as 30%, 50% and 70% of
the total polymer mass, respectively. The system was then
treated with 2 ml 1% EDAC solution at 37 °C for 5 min.
After maintaining the solution at 70 °C for 5 min to elimi-
nate the entrapped air bubbles, it was frozen at 25 °C for
2 h and lyophilized at 50 °C for 48 h. The dried scaffold
was further crosslinked by 30 ml 0.3% EDAC at 37 °C
for 1 h and then lyophilized for 24 h.
2) EDAC crosslinking
3) Lyophilization
c
330 H. Tan et al. / Acta Biomaterialia 5 (2009) 328–337
2.4. Structure observation
For microstructure observation, the scaffolds were dehy-
drated by treatment with a series of graded ethanol solu-
tions (75%, 85%, 95% and 100%) at room temperature,
each for 15 min, and then treated in a vacuum oven at
50 °C overnight. The scaffolds were then cut into pieces
with a razor blade before coating with gold for scanning
electron microscopy (SIRION 100, FEI) examination.
2.5. Porosity and density
To determine the porosity (P) of the scaffolds, volumes
of the scaffolds (Vs) and pores (Vp) were measured. Vs
was measured from the scaffold geometry (length, width
and height). Vp was calculated by an ethanol infiltration
method. Briefly, the weighed scaffolds (W0) were incubated
in absolute ethanol at room temperature. The system was
maintained for 15 min in a desiccator under reduced pres-
sure to remove air bubbles. The scaffolds were taken out
and wiped superficially with a filter paper to remove the
surface ethanol, and then weighed immediately (We). Vp
was defined as (We W0)/qe, where qe (0.789 mg ml–1) rep-
resents the ethanol density at room temperature. The
porosity of the scaffolds was calculated according to
P = Vp/Vs  100%. The apparent density of the scaffolds
was defined as W0/Vs.
2.6. Compressive property
Cylindrical scaffold samples (20 mm diameter  18 mm
high) were compressed by a mechanical tester (Zwick
Roell, Germany). The cross-head speed was set at
1 mm min 1. The compressive modulus was determined
from the compressive curve in the initial strain range of
2–6%. Hydrated samples were measured immediately after
they were incubated in phosphate-buffered saline (PBS, pH
7.4) at 37 °C for 30 min.
2.7. Weight loss
The weight loss of the scaffolds was monitored as a func-
tion of incubation time in PBS at 37 °C. Briefly, 10 mg of
each of the scaffolds was incubated in 2 ml PBS at 37 °C in
a waterbath. The incubated PBS was replaced by fresh PBS
every 2 days. No significant change in pH was found during
this incubation because of the relatively slow degradation of
PLGA. The scaffolds were taken from the PBS at the desired
time intervals, washed with water, lyophilized and weighed
(Wt). The weight loss ratio was defined as (W0 Wt)/
W0  100%. Each experiment was repeated three times,
and the value is reported as means ± SD (n = 3).
2.8. PBS uptake
To determine the PBS uptake property, the completely
dried scaffolds were incubated in PBS at 37 °C. The
hydrated scaffolds were taken out at the desired time inter-
vals, wiped superficially with a filter paper to remove the
surface water, weighed (Wwt) and then lyophilized (Wdt).
The uptake ratio was defined as (Wwt Wdt)/Wdt. The
value is expressed as means ± SD (n = 3).
2.9. Cell culture
Chondrocytes were isolated from cartilage tissue of rab-
bit ears (New Zealand white) under institutional guidelines
and were routinely cultured [15,29]. Briefly, the cartilage
tissue was cut into small pieces. The chondrocytes were iso-
lated by incubating the cartilage pieces in Dulbecco’s mod-
ified Eagle’s medium (DMEM) containing 0.2%
collagenase type II at 37 °C for 5 h under agitation. The
isolated chondrocytes were centrifuged, resuspended in
DMEM supplemented with 10% FBS, 100 U ml 1 penicil-
lin and 100 lg ml–1 streptomycin. The cells were then
seeded in an 11 cm plastic tissue culture dish (Falcon)
and were incubated in a humidified atmosphere of 95%
air and 5% CO2 at 37 °C. After a confluent cell layer was
formed, the cells were detached using 0.25% trypsin in
PBS and were resuspended in PBS, and used for the
experiments.
The scaffolds were cut into 6  6  3 mm3 pieces and
sterilized with a 75 vol.% ethanol solution. After washing
in PBS, all the scaffolds were placed in the bottoms of a
24-well tissue culture plate. Chondrocyte suspension
(300 ll) with a cell density of 500  104 cells ml–1 was
injected into each scaffold using a 1 ml syringe (inner diam-
eter 0.5 mm). The cells were alive when seeded as deter-
mined by the Trypan Blue dye test. The cell–scaffold
constructs were cultured statically in DMEM/10% FBS
culture medium for a given time. The culture conditions
were the same as described above.
2.10. Cell viability and proliferation
Cell viability was measured by MTT (3-(4,5-
dimethyl)thiazol-2-yl-2,5-dimethyl tetrazolium bromide)
assay during cell culture. After supplementation with
200 ll MTT solution (5 mg ml 1), the cells were continu-
ally cultured for another 5 h. During this period, viable
cells could reduce the MTT to formazan pigment, which
was dissolved by 800 ll dimethyl sulfoxide (DMSO) after
removal of the culture medium. The absorbance at
490 nm was recorded by a microplate reader (Bio-Rad
550).
The cell number in the samples was assessed by quanti-
fying DNA (7.7 pg DNA/cell) in the constructs using Hoe-
chst 33258 dye (Sigma) assay [30,31]. Samples were frozen
at 20 °C for 2 h, and lyophilized for 24 h. Each sample
was then digested with 1 ml 1% w/v papain/0.09% diso-
dium ethylenediaminetetraacetic acid (Na2EDTA). Shortly
after, 100 ll of the papain-digested solution was added into
2 ml Hoechst 33258 solution (1 lg ml 1), the fluorescence
intensity at 450 nm was measured by a fluorescent spectro-
 H. Tan et al. / Acta Biomaterialia 5 (2009) 328–337
photometer (RF-5301PC, Shimadzu). The content of DNA
in the samples was determined by referring to a calibration
curve of standard DNA (calf thymus DNA).
2.11. Cell distribution and morphology
Chondrocyte distribution in the scaffolds was observed
under confocal laser scanning microscopy (LSM510, Zeiss)
after the cells were cultured for 7 and 14 days. The scaffolds
were taken out from the culture plate and rinsed with PBS
gently, then immerged into 5 lg ml 1 FDA/PBS solution
and incubated for 10 min at room temperature. Using this
fluorescent staining, only the viable cells in the scaffolds
can be visualized under CLSM. The cells in the scaffolds
were further observed under SEM after fixation with
2.5% glutaraldehyde at 4 °C for 24 h. The cell-scaffold con-
structs were sequentially treated in a series of ethanol solu-
tion. The constructs were then treated with acetone and
isoamylacetate successively, each for 15 min at room tem-
perature. Finally, critical point drying was performed,
and the constructs were observed by SEM (Stereoscan
260, Cambridge) after coating with a thin gold layer.
2.12. GAGs secretion assay
The total contents of sulfated glycosaminoglycans
(GAGs) secreted by the chondrocytes in the scaffolds were
determined quantitatively using a 1,9-dimethylmethylene
blue method with chondroitin sulfate as a standard [32].
Briefly, the constructs were freeze-dried after the chondro-
cytes had been cultured for 7, 14 and 21 days. The dried
scaffolds were digested with papain in a buffer of 0.1 M
KH2PO4, 5 mM Na2EDTA and 5 mM cysteine–HCl at
pH 6.0 and 60 °C for 6 h. The dye solution was prepared
Fig. 1. SEM images of (a) PLGA microspheres, (b) CS, (c), (d) and (f) are 30%MS, 50%MS and 70%MS, respectively, (e) is a magnified image of (d).
331
by dissolving 16 mg of 1,9-dimethylmethylene blue in 1 l
distilled water containing 3.04 g glycine, 2.37 g NaCl and
95 ml 0.1 M HCl. 1,9-Dimethylmethylene blue solution
(2 ml) was then added to 100 ll of the papain-digested
solution. After 5 min, the absorbance was measured at
525 nm by UV–visible spectroscopy (UV-Probe 2550,
Shimadzu).
2.13. Statistical analysis
Experimental data were analyzed using ANOVA. The
significance level was set as P < 0.05. Results are reported
as means ± SD.
3. Results
3.1. Scaffold morphology
Morphologies of the PLGA microspheres, gelatin/chito-
san/hyaluronan ternary complex scaffold, and PLGA
microsphere-integrated scaffolds were observed by SEM
(Fig. 1). Under the fabrication conditions used here, the
PLGA microspheres have a spherical morphology with a
smooth surface, and are less than 50 lm (mostly within
5–40 lm) in diameter (Fig. 1a). The cross-sectional porous
microstructure of the gelatin/chitosan/hyaluronan ternary
complex scaffold (control scaffold, CS) has an average pore
diameter of 200 lm (Fig. 1b). The pores are evenly distrib-
uted throughout the scaffold. Fig. 1c, d and f reveal the
morphologies of the 30%, 50% and 70% PLGA micro-
sphere-integrated scaffolds (30%MS, 50%MS and
70%MS), respectively. The PLGA microspheres are distrib-
uted homogeneously throughout all the scaffolds. Both
30%MS and 50%MS show interconnected pores with a
332 H. Tan et al. / Acta Biomaterialia 5 (2009) 328–337

mean diameter of 200 lm. Compared with 30%MS, there 3.3. Compressive properties
are more microspheres residing in the skeletons of the
50%MS (Fig. 1e). In contrast to this, integrating 70% Fig. 3a shows the stress–strain curves of the scaffolds in
PLGA microspheres leads to the disappearance of much a hydrated state. Compared with the control scaffold, all
of the porous structure (Fig. 1f). The remaining pores are the PLGA microsphere-integrated scaffolds have a larger
mostly isolated from each other. stress vs. strain, and a higher content of the microspheres
resulted in a larger stress at the same strain. The compres-
3.2. Porosity and density sive modulii were calculated from the stress–strain curves
when the strain was lower than 6% (the elastic region)
Fig. 2 presents the porosity and density of the composite (Fig. 3b). The value increased from 0.07 MPa (CS) to
scaffolds. The control scaffold has a porosity of 93%, 0.22 MPa (30%MS), significantly to 0.53 and 0.68 MPa
which is slightly smaller than that of the theoretical value for the 50%MS and 70%MS, respectively. The morphology
(95%) obtained from its solid content. Integration of of the completely compressed scaffold containing 50%
the PLGA microspheres has caused a decrease in the microspheres is shown in Fig. 4. This shows that in both
porosity. However, even integrating 50% PLGA micro- dry and hydrated states the pores were collapsed and the
spheres still gives a porosity of over 90%. When the micro- pore walls contacted each other. More PLGA microspheres
sphere content reached 70%, however, the porosity sharply were exposed.
decreased to 83%. The apparent density shows a reverse
tendency with the porosity, i.e. a higher microsphere con- 3.4. Weight loss
tent yields a scaffold with a higher density.
Fig. 5 shows a gross view of the scaffolds after incuba-
tion in PBS at 37 °C for different times. The initially
hydrated control scaffold (CS) had a transparent appear-
0.20
95 ance, whereas the PLGA-integrated scaffolds were opaque.
As the incubation proceeded, the shape of all the scaffolds
changed progressively. After 21 days of incubation, the
0.16
Density (mg/mm )

50%MS and 70%MS showed a stronger ability to preserve

3
Porosity (%)

90 their original shapes than the control and the 30%MS,

Porosity 0.12
though macroscopic deformation and defects could still
Density be observed.
85
Profiles of the weight loss of the scaffolds are shown in
0.08 Fig. 6. All scaffolds lost weight almost linearly with incuba-
tion time. No significant difference was found between the
control and the 30%MS except for days 11 and 14, where a
80 0.04 slower weight loss of the 30%MS was observed. Although
0 10 20 30 40 50 60 70 no significant difference was found either for the 50%MS
PLGA (%) and 70%MS before days 11 and 7, respectively, signifi-
Fig. 2. Porosity and density of the PLGA microsphere-integrated scaffold cantly slower loss rates (P < 0.05) were indeed found after
as a function of microsphere weight ratio. day 11. At 14 days, the weight loss for the CS, 30%MS,

a 0.4
CS
b 0.9
Compressive modulus (MPa)

30% MS *
50% MS
70% MS
0.3 *
Stress (N/mm2)

0.6

0.2

0.3
0.1

0.0
0.0
0 15 30 45 60 CS 30% 50% 70%
Strain (%) PLGA

Fig. 3. (a) Stress–strain curves of the PLGA microsphere-integrated scaffold with different PLGA microsphere contents in a wet state. (b) Compressive
modulus derived from (a).
 H. Tan et al. / Acta Biomaterialia 5 (2009) 328–337 333

Fig. 4. SEM images of the 50%MS after total compression in (a) a dry and (b) a hydrated state.

Fig. 5. Gross appearances of CS, 30%MS, 50%MS, and 70%MS after incubation in PBS at 37 °C for different times (1 h, 7, 14 and 21 days). Scale bar,
1 cm.

50%MS and 70%MS was 26%, 17.2%, 13.3% and 10.1%, after PLGA microsphere integration. However, these
respectively. reduced rates were more insensitive to incubation time.

3.5. PBS uptake property 3.6. Chondrocyte culture results

The PBS uptake ability of all the scaffolds decreased
with incubation time, and a faster rate of decrease was
found for the control scaffold before day 4 (Fig. 7). The ini-
tial PBS uptake ratio at 1 h for the CS, 30%MS, 50%MS
and 70%MS was 17.4, 10.9, 8.6 and 6.4, respectively. These
values decreased to 9.9, 7.4, 6.2 and 3.9 at day 7, and fur-
ther to 8.7, 5.9, 5.1 and 3.2 at day 14, respectively. The
overall PBS uptake capacity was significantly decreased
In this study, 50%MS was compared with the control in
terms of chondrocyte viability and number, and GAGs
production. Fig. 8a shows that the cytoviability increased
monotonously along with the culture time in vitro. DNA
assay recorded a consistent increase in the cell number.
No significant difference was found between the control
and the 50%MS at each time interval (P > 0.05). The cell
number in the 50%MS was 114  104 and 138  104
334 H. Tan et al. / Acta Biomaterialia 5 (2009) 328–337

30 7 days of culture the chondrocytes primarily presented in

CS *
30% MS the superficial area of the scaffold and maintained their
50% MS round morphology. After 14 days of culture, the chondro-
70% MS *
cytes distributed more densely (Fig. 9b), and contacted
Weight loss (%)

20
with each other to form larger aggregates on the scaffold.
This merging of the cells indicates the presence of ECM.
After staining with FDA, the viable cells in the scaffolds
10 were imaged by CLSM (Fig. 10). A number of PLGA
microspheres in the scaffold can be identified as indicated
by the arrows at 7 days (Fig. 10a), but most of them were
covered by the cells after 14 days (Fig. 10b). The chondro-
0 cytes in the control scaffold had exactly the same morphol-
0 2 4 6 8 10 12 14 ogy, and so the images are not shown.
Time (d) The amounts of GAGs secreted by the chondrocytes
after 7, 14 and 21 days in the control and the 50%MS is
Fig. 6. Weight loss of the PLGA microsphere-integrated scaffold as a
function of incubation time in PBS at 37 °C. compared in Fig. 11. GAG amount significantly increased
with culture time (P < 0.05), but there was no significant
difference (P > 0.05) between the two scaffolds at each time
20 interval.
* CS
* 30% MS
50% MS 4. Discussion
70% MS
15 *
PBS uptake ratio

* Biodegradable scaffolds play key roles in regulating cell

* *
* adhesion, proliferation, differentiation and quality of the
10 * regenerated tissues, and are thus critical in the field of tis-
sue engineering and regenerative medicine. In this work,
a composite scaffold was made by integrating PLGA
5
microspheres into a ternary scaffold composed of gelatin,
chitosan and hyaluronan. Unlike its counterparts of evenly
mixed PLGA and biomacromolecules, here PLGA was
0
0 2 4 6 8 10 12 14 incorporated into the biomacromolecule scaffold in the
Time (d) form of microspheres. This strategy produced a scaffold
with both enhanced mechanical strength and good biocom-
Fig. 7. Uptake capacity of PLGA microsphere-integrated scaffold as a
function of incubation time in PBS at 37 °C.
patibility. In addition to providing bioactive sites for cell
anchorage, the mechanical and anti-degradable properties
of this scaffold were improved to meet the requirements
after 24 h and 7 days of culture, respectively, and this sig- of chondrogenesis.
nificantly increased to 236  104 after 14 days of culture In order to essentially retain the original microstructure
(P < 0.05). of the ternary scaffold, PLGA microspheres <50 lm were
SEM images of the 50%MS seeded with chondrocytes used. SEM observations demonstrated that the micro-
are presented in Fig. 9. As shown in Fig. 9a and c, after spheres in the 30%MS and 50%MS (Fig. 1c and d) were

a 1.0
CS b 300
CS
50% MS 50% MS *
Cell number ( x104 )

0.8 200
Abs

0.6 100

0.4 0
3 6 9 12 15 18 21 24h 7d 14d
Time (d) Time

Fig. 8. (a) MTT viability of chondrocytes in CS and 50%MS as a function of culture time. (b) Chondrocyte number in CS and 50%MS at culture times of
24 h, 7 and 14 days. Each well was seeded with 300 ll cells with a density of 500  104 cells ml 1.
 H. Tan et al. / Acta Biomaterialia 5 (2009) 328–337 335

Fig. 9. SEM images of 50%MS in which chondrocytes were cultured for (a) 7 days and (b) 14 days. (c) and (d) are magnified images of (a) and (b),
respectively. Each well was seeded with 300 ll cells with a density of 500  104 cells ml 1.

Fig. 10. CLSM images (FDA staining) to show the viable chondrocytes cultured in 50%MS for (a) 7 days and (b) 14 days. Each well was seeded with
300 ll cells with a density of 500  104 cells ml 1.

distributed evenly in the pore walls, whereas the pore struc-
ture was perfectly preserved. A still higher amount of
microspheres significantly improves the viscosity of the sys-
tem, and thus retards molecular transplantation during the
freezing process, i.e. a process of phase separation. In such
a case, the same quenching condition yields a scaffold with
smaller pores [5]. On the other hand, the increased solid
content also favors formation of a dense scaffold with smal-
ler pores. Therefore, for practical applications, the micro-
sphere content should be controlled below 50%, so that
the resulting scaffolds have large enough pores (200 lm)
and porosity (>90%). These features are advantageous
for cell infiltration and proliferation, and for mass trans-
portation [33].
Mechanical properties, especially compressive strength,
are particularly important for scaffolds used for chondro-
genesis, since they are closely associated with the shape-
persistency in practical operations and applications. We
emphasize here the mechanical performance in a hydrated
state since the scaffolds and constructs are used exclusively
336
1.0
CS
50% MS
GAG content (mg/cm3 scaffold)
0.8
0.6
0.4
0.2
7d 14d
Time
Fig. 11. Amount of GAGs synthesized by chondrocytes seeded in CS and
50%MS for 7, 14 and 21 days. Each well was seeded with 300 ll cells with
a density of 500  104 cells ml 1.
in contact with culture medium in vitro or body fluid in
vivo. The microsphere integration accounts for the
improvement in the compressive stress and the compressive
modulus of the composite scaffold. For example, the com-
pressive modulus was improved by a factor of 8 when 50%
microspheres were integrated (Fig. 3b). In addition to
toughening the scaffold directly, the microspheres can also
partially maintain the porous structure even in a collapsed
state (Fig. 4). It is conceivable that there is still space in
between the microspheres even though the scaffold is com-
pletely compressed [29]. In fact, the theoretical closest com-
pact factor for spheres is 0.74, implying that at least 26%
space is left in the piled microspheres [34].
The weight loss and the medium uptake ability, which
affect the durability and the nutrition and waste transpor-
tation, are two other factors that influence the performance
of the scaffolds. Results in Figs. 6 and 7 show that the fas-
ter weight loss of the control scaffold was significantly slo-
wed down by integrating the PLGA microspheres, whereas
the medium uptake ability remained sufficiently high. The
weight loss and medium uptake property depend on both
the hydrophilicity and the three-dimensional structure of
the scaffold. The PLGA microspheres are more hydropho-
bic and less sensitive to hydrolyzation than the natural bio-
macromolecules, especially gelatin and hyaluronan.
Therefore, the scaffolds containing the PLGA microspheres
show a stronger resistance to weight loss than that of the
control ternary scaffold, and this intensity is enhanced at
higher microsphere content. The weakening of the PBS
uptake ability after PLGA microsphere integration is
attributed to the hydrophobic increase and the total weight
increase of the scaffold. The slower decrease of the uptake
ability of the PLGA microsphere-integrated scaffolds
reveals that the three-dimensional persistency of the scaf-
folds is better than that of the control one.
In summary, the PLGA microsphere-integrated scaf-
folds have better comprehensive physical properties com-
pared with the ternary complex scaffold, in particular
H. Tan et al. / Acta Biomaterialia 5 (2009) 328–337
when the microsphere content is 50%. Therefore, this scaf-
fold was further subject to evaluation of its biological per-
* *
formance by chondrocyte culture in vitro.
The MTT and DNA analyses demonstrate that the
50%MS has cell viability as good as that of the control
scaffold. In both kinds of scaffolds, chondrocytes can nor-
mally proliferate and produce ECM such as GAGs, dem-
onstrating that the good biocompatibility of the initial
ternary scaffold has not been deteriorated after integration
of the PLGA microspheres. GAG is a kind of polysaccha-
ride, which together with collagen type II forms the main
components of the cartilage matrix. Secretion of GAGs
21d by in vitro cultured chondrocytes can be regarded as a sign
that the cell phenotype is maintained. Although there is no
significant difference between the two kinds of scaffolds, the
total biosynthesized GAGs in both scaffolds is significantly
increased with the culture time. Here, due to interference of
the gelatin, a structural analog of collagen, collagen type II
is not detected in the constructs. Moreover, the amount of
the synthesized matrix is not yet enough to form a cartilage
analog. Further studies are underway to culture the con-
structs for a longer period and in vivo.
It is worth mentioning that bioactive substances such as
cell growth factors, basic fibroblast growth factor (bFGF)
for example, can also be loaded into the polymer micro-
spheres. However, our results showed that the bioactivity
of the bFGF was largely lost although the loading could
be successfully achieved. It is possible that in the future,
loading of substances with low sensitivity to organic sol-
vents will be more promising. Further in vivo experiments
are required to answer the long-term effects of the scaffolds,
assuming the matrix has been removed, leaving only the
PLGA microspheres. Nevertheless, we can still envisage
such a case. On the one hand, the macroscopic shape
should change significantly, on the other hand, the newly
formed ECM may have replaced the foreign matrix. The
existence of the PLGA microspheres would be beneficial
to the maintaining the topology because of the lower deg-
radation speed. This may also temporarily contribute to
cartilage regeneration.
5. Conclusions
Composite scaffolds composed of PLGA microspheres
and gelatin, chitosan and hyaluronan were fabricated.
The composite scaffolds have larger compressive modulii,
lower weight loss, good porosity (>90%) and medium
uptake properties, and sufficiently large pore size
(200 lm) when the PLGA microsphere content is less
than 50%. In vitro chondrocyte culture reveals that the
50% PLGA-integrated scaffold has good cell attachment
(24 h), viability, proliferation and GAG secretion, and is
not significantly different in these respects to the control
gelatin/chitosan/hyaluronan scaffold. All these results dem-
onstrate that the PLGA microsphere-integrated scaffolds,
especially the one with 50% PLGA microspheres, have bet-
ter physical performance and preserved biocompatibility to
 H. Tan et al. / Acta Biomaterialia 5 (2009) 328–337
support chondrocyte growth, and thus have greater poten-
tial to be used for chondrogenesis.
Acknowledgments
This study is financially supported by the National
High-tech Research and Development Program
(2006AA03Z442), the Major State Basic Research Program
of China (2005CB623902), the Science and Technology
Program of Zhejiang Province (2006C13022), and the Na-
tional Science Fund for Distinguished Young Scholars of
China (No. 50425311).
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