Materials Letters 61 (2007) 2601 – 2605

www.elsevier.com/locate/matlet

Preparation and characterization of collagen-modified

polylactide microparticles
Yumei Xiao a , Yazhen Xu b , Jian Lu a , Xiangdong Zhu a , Hongsong Fan a,⁎, Xingdong Zhang a
a
Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China
b
Shanghai Institute of Materia Medica Chinese Academy of Sciences, Shanghai 201203, China
Received 8 April 2006; accepted 4 October 2006
Available online 27 October 2006

Abstract

To improve cytocompatibility of polylactide (PLA) and to obtain an injectable scaffold for tissue engineering, collagen-modified PLA (CPLA)
microparticles were prepared. Poly-(α-methacrylic acid)-grafted PLA (PMAA-PLA) was obtained by photooxidization and UV induced
polymerization. Suspension of PMAA-PLA microspheres with an average size of 172.8 ± 3.6 nm was prepared with solvent evaporation technique.
CPLA microparticles were prepared by adding collagen acetic acid solution into PMAA-PLA microsphere suspension prepared above. FTIR
spectrum of PMAA-PLA confirmed that PMAA had been grafted on PLA surface. Analytical results of FTIR, XPS, SEM, hematoxylin and eosin
(HE) stained and zeta potential measurement showed that the CPLA microparticles obtained by modifying PMAA-PLA microspheres with
collagen molecules uniformly have a microporous structure and a particle size of less than 100 μm. The CPLA microparticles were expected to be
used as an injectable scaffold for tissue regeneration.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Microparticles; Polylactide; Collagen; Microspheres; Surface modification

1. Introduction PLA (CPLA) microparticles were prepared by adding collagen

Tissue engineering is a promising approach to repair damaged
tissues/organs such as cartilage, skin and bone, in which a number
of synthetic polymers have been used as scaffold materials.
Polylactide (PLA) is one of the most common polymers due to its
advantages such as nontoxicity, processibility and biodegradabil-
ity. However, its cytocompatibility needs to be improved because
its hydrophobicity retards cell attachment [1,2]. One approach to
improve its cytocompatibility is to introduce collagen onto the
surface of PLA through surface modification because collagen
can specifically bind with integrins on cell membrane, which can
effectively accelerate cell attachment and spreading [3–10].
Injectable scaffold has attracted more and more research in-
terests among tissue engineering scaffolds because of its easiness
of being transplanted [10–13]. In this study, collagen-modified
⁎ Corresponding author. Tel.: +86 28 85410703; fax: +86 28 85410653.
E-mail address: xymzl2000@126.com (H. Fan).
0167-577X/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.matlet.2006.10.006
solution into PMAA-PLA microsphere suspension. The obtained
CPLA microparticles are expected to be used as an injectable
scaffold for tissue regeneration.
2. Materials and methods
2.1. Preparation of CPLA microparticles
Poly (α-methacrylic acid) (PMAA) was introduced onto the
PLA surface using a grafting polymeric method as described
previously [14]. The PLA particles (medical grade, purchased
from Dikang Biomedical Co., Ltd, China) were immersed in
hydrogen peroxide solution (30%, AR) and irradiated with UV
light for 50 min to introduce –OOH groups onto the surface of
PLA particles. The photooxidized particles were rinsed with
deionized water to remove excess hydrogen peroxide and then
immersed into α-methacrylic acid (MAA, AR) aqueous solution
(15%). PMAA-grafted PLA (PMAA-PLA) was obtained by
grafting polymerization under UV irradiation for 30 min. PMAA-
2602 Y. Xiao et al. / Materials Letters 61 (2007) 2601–2605

PLA was then rinsed with deionized water at 65 °C to remove

homopolymers [2] and dried.
The PMAA-PLA acetone solution was added into Na2HPO4
aqueous solution (pH = 7–8) with stirring. The suspension was
stirred for about 4 h at 37 °C to remove acetone in the solution
and neutral PMAA-PLA microsphere suspension was obtained.
Then, collagen acetic acid solution was dropped into PMAA-
PLA microsphere suspension until much flocculent precipita-
tion appeared. The obtained CPLA precipitates were washed
with deionized water thoroughly, dried at room temperature and
ground. Collagen acetic acid solution (medical grade) was
added into Na2HPO4 aqueous solution (pH = 7) as control. And
acidic solution was dropped into the neutral PMAA-PLA
microsphere suspension until flocculent PMAA-PLA deposit
appeared. Then the obtained PMAA-PLA deposit was rinsed
with deionized water and dried. Fig. 1. The size distribution on curves of PMAA-PLA microspheres (3
measurements).
2.2. Characterization
microsphere suspension presented negative value, which meant that
The average size and zeta potential of the PMAA-PLA PMAA-PLA microspheres in the suspension had net negative surface
microspheres were measured by a Nano ZS instrument charge. When acidic collagen solution with positive surface charge was
(Malvern Co., UK). Data for each sample were obtained from added into PMAA-PLA microsphere suspension, the positive charge
on collagen molecule surface would neutralize negative charge on
3 measurements. FTIR spectra were obtained using Fourier-
PMAA-PLA microsphere surface. It could be assumed that collagen
transform infrared spectroscopy (PE spectrum one (B)). molecules congregate on the PMAA-PLA microsphere surface due to
Scanning electron microscopy (SEM, JSM5900) was used to electrostatic interaction and other interactions between them. The
observe the size and surface morphology of the obtained PMAA-PLA microspheres in the suspension didn't flocculate because
materials. X-ray photoelectronic spectra were recorded employ- they had high negative zeta potential and tended to repel each other.
ing MgKα excitation radiation by an X-ray photoelectronic When there was no force to prevent the particles from coming together
spectroscopy (XSAM 800). and flocculating, PMAA-PLA microspheres congregated collagen
The surface modification of PMAA-PLA microspheres was molecules would occur conglomerating into CPLA deposit.
confirmed further by staining collagen in CPLA microparticles Fig. 2 showed the relationship between zeta potential and pH value
with hematoxylin and eosin (HE). CPLA microparticles were of PMAA-PLA microsphere suspension. From Fig. 2 it could be seen
embedded in paraffin and sections of about 10 μm were cut. The that the absolute value of zeta potential of PMAA-PLA microsphere
suspension diminished along with decreases of pH value of the
sections were dewaxed with dimethylbenzene and stained with
suspension system. When the pH value diminished to 5, PMAA-PLA
HE for visualization of the modified collagen under light
microsphere suspension still had high negative zeta potential and there
microscope [15]. And acetone solution of PMAA-PLA used as was no deposit appearing. Only when the pH decreased to 3.0, the
control was dropped on the cover slip, stained with HE after the absolute value of zeta potential of PMAA-PLA microsphere suspen-
solvent evaporation, and observed under microscope. sion was less than 30 mV and flocculent PMAA-PLA deposit could be
observed. In addition, after collagen acetic acid solution was added into
3. Results and discussion the Na2HPO4 aqueous solution (pH = 7), there was no deposit
appearing at any time. However, in our study, flocculent CPLA deposit
3.1. The measurement of particle size and zeta potential appeared at pH ≈ 5. After CPLA deposit produced in acetic acid
solution (pH ≈ 5) was washed with deionized water thoroughly,
The average particle size and zeta potential of the PMAA-PLA collagen was still found in CPLA microparticles. All these results
microspheres in the suspension obtained were shown in Table 1. The
size of PMAA-PLA microspheres was from 59 nm to 615 nm (Fig. 1).
The absolute value of zeta potential of PMAA-PLA microsphere
suspension was much higher than 30 mV, which meant that the PMAA-
PLA microspheres in the suspension were not readily conglomerated
[16]. Therefore, the PMAA-PLA microsphere suspension with an
average particle size of 172.8 ± 3.6 nm could stably exist without any
deposit appearing. At the same time, zeta potential of PMAA-PLA

Table 1
The average size and zeta potential for PMAA-PLA microspheres
Size, nm Zeta potentials, mV
172.8 ± 3.6 −95.0 ± 0.6
Fig. 2. The effect curve of zeta potential on the pH value.
 Y. Xiao et al. / Materials Letters 61 (2007) 2601–2605
implied that there were strong interactions other than physical
adsorption between PMAA-PLA microsphere surface and collagen
molecules in CPLA microparticles. Therefore, CPLA microparticles
could be obtained by the method used in this study.
3.2. FTIR spectra analysis
The FTIR spectra of PLA, PMAA-PLA, PMAA-PLA treated with
sodium hydroxide solution and CPLA were shown in Fig. 3a–d. From
the FTIR spectra it could be seen that the peaks of stretching vibration
attributed to O–H in carboxyl groups and C_O at 3583 cm− l and
1755 cm− l in PMAA-PLA were stronger than those of PLA, and the
width of C_O peak of PMAA-PLA was bigger than that of PLA,
which indicated that PMAA was grafted onto the PLA surface [14].
After alkaline treatment of PMAA-PLA, the peak of absorbance at
3583 cm− l disappeared and a new peak that attributed to the
absorbance of carboxylate groups (–COO−) [17,18] appeared at
1414 cm− l (Fig. 3c). This showed that the carboxyl groups (–COOH)
on PMAA-PLA surface were easily ionized into –COO− in alkaline
aqueous solution [14]. Moreover, it was reported that some carboxyl
groups on PMAA-grafted PLA film were transferred to –COO− [2].
Therefore, PMAA-PLA microsphere surface in the suspension
containing Na2HPO4 should have negative charge which was
according with the result of zeta potential measurement. FTIR
spectrum of CPLA (Fig. 3d) showed the characteristic peaks of
collagen. The peak at about 3500–3300 cm− 1 arose from amide A band
due to N–H stretching vibration. The peak at 2947 cm− 1 was ascribed
to amide B band and stretching vibration of –CH3 of PLA. The peak of
amide I band that appeared at 1656 cm− 1 was attributed to the
stretching vibrations of the C_O groups in collagen [19]. And the
peak at 1547 cm− 1 arose from the N–H bending vibration coupled to
the C–N stretching vibration of protein amide groups. These results
indicated the presence of collagen in CPLA microparticles, which
would promote cell attachment and spreading [3–6].
Fig. 3. FTIR spectra of (a) PLA, (b) PMAA-PLA, (c) PMAA-PLA treated with
sodium hydroxide solution and (d) CPLA.
2603
Fig. 4. XPS spectra of (a) CPLA and (b) PLA.
3.3. XPS analysis
Nitrogen atom was present in collagen molecules but not in PLA and
PMAA-PLA. Therefore, a convincing evidence of collagen introduced
onto PMAA-PLA microsphere surface was the appearance of the peak
of nitrogen in the XPS observation of CPLA microparticles [2,5]. In our
present study, a peak of nitrogen was detected from the CPLA
microparticle surface while no peak of nitrogen was found in the control
group (Fig. 4), which indicated that collagen had been introduced onto
the PMAA-PLA microsphere surface by the above method.
3.4. SEM analysis and HE staining
SEM photos showed the surface morphology of PMAA-PLA
microspheres, PMAA-PLA deposit (obtained at pH = 2.7) and CPLA
microparticles (Fig. 5). In PMAA-PLA microsphere suspension,
PMAA-PLA microspheres had a spherical morphology. The diameter
of PMAA-PLA microspheres ranged from 50 nm to 600 nm according
to the result of particle size measurement with Nano ZS instrument.
After collagen solution was added into PMAA-PLA microsphere sus-
pension, PMAA-PLA microspheres occurred congregating through
interactions with collagen molecules and came into being irregular
CPLA microparticles with microporous structure and particle size of
less than 100 μm. Comparing the SEM photos of CPLA microparticles
and PMAA-PLA deposit, it could be seen that the surface morphology
of CPLA microparticles and PMAA-PLA deposit was completely
different. PMAA-PLA deposit was composed of many PMAA-PLA
microspheres or anamorphic PMAA-PLA microspheres but CPLA was
not. In CPLA microparticles, no same surface morphology as PMAA-
PLA deposit could be observed under SEM. Therefore, it could be
concluded that CPLA microparticles were not a mixture of PMAA-
PLA microspheres and collagen particles but collagen-modified PLA
microparticles. The obtained CPLA microparticles had a potential to be
used as injectable scaffold in tissue engineering.
CPLA microparticles were stained lightly by HE due to collagen
addition while PMAA-PLA film did not show any staining with HE
(Fig. 6). Staining for CPLA microparticles further confirmed the
presence of collagen component in CPLA microparticles. Further study
on the cytocompatibility in vitro and injectability in vivo of CPLA
microparticles is needed.
2604 Y. Xiao et al. / Materials Letters 61 (2007) 2601–2605

Fig. 5. Surface morphology of (a) PMAA-PLA microspheres, (b) CPLA microparticles and (c) PMAA-PLA deposit.

Fig. 6. Optical micrographs of HE staining (a) CPLA and (b) PMAA-PLA.

 Y. Xiao et al. / Materials Letters 61 (2007) 2601–2605
4. Conclusion
Carboxyl groups were introduced onto inert PLA surface
through UV induced grafting of PMAA. Subsequently collagen-
modified PLA microparticles with microporous structure were
successfully prepared through interactions between PMAA-
grafted PLA microspheres and collagen molecules. FTIR, XPS,
SEM, HE stained and zeta potential measurement confirmed the
occurrence of modification of collagen on PLA surface. These
collagen-modified PLA (CPLA) microparticles are supposed to
support the attachment and proliferation of cells better, which
might be used as injectable scaffold for tissue engineering.
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