Immobilization of Natural Macromolecules on Poly-L-Lactic Acid
Membrane Surface in Order to Improve Its Cytocompatibility
Zuwei Ma, Changyou Gao, Yihong Gong, Jian Ji, Jiacong Shen
Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
Received 17 January 2002; revised 25 June 2002; accepted 26 June 2002
Published online 00 Month 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.10470
Abstract: With the use of a grafting– coating method, three kinds of natural macromolecules, that is, gelatin, collagen, or chitosan, were immobilized on poly-L-lactic acid (PLLA)
membrane surfaces with the goal of improving of cellular interactions. Attenuated total
reflectance infrared spectroscopy (ATR-IR), x-ray photoelectron spectroscopy (XPS) and
surface morphology analysis using scanning electronic microscopy (SEM) confirmed that the
natural macromolecule layers adhered tightly to the hydrophobic PLLA membrane surfaces.
Chondrocyte culture showed that the modified PLLA membranes had higher cell attachment,
higher cell proliferation rate, and higher cell activity than the control PLLA membrane.
Moreover, the chondrocytes were more spread out on the modified PLLA membranes than on
the control PLLA membranes. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res (Appl Biomater) 63:
838 – 847, 2002
Keywords: poly-L-lactic acid; surface modification; biocompatibility; natural macromolecules; chondrocyte
INTRODUCTION
Synthetic biodegradable polymers, such as polylactic acid
(PLA), polyglycolic acid (PGA), and polylactic-co-glycolic
acid (PLGA) are widely used to build three-dimensional
scaffold for generation of tissue-engineered organs due to
their good biodegradability, good mechanical properties, and
proper degradation rate, which is often comparable to the
healing time of the damaged human tissues. Transplantation
of isolated cells seeded in a synthetic biodegradable scaffold
has been investigated as a mean of producing biologic substitutes to regenerate or replace the damaged tissues such as
articulate cartilage.1
However, most synthetic materials do not possess the
surface properties necessary for tissue engineering scaffold.
For example, the surface of poly-L-lactic acid (PLLA) is
hydrophobic and does not promote cell adhesion.2 It is the
surface of a biomaterial that first comes into contact with the
living body, so the initial response of the cells to the biomaterial must depend on the surface properties. Therefore, the
most convenient method to improve the cytocompatibility of
PLLA is to introduce a cytocompatible layer on the polymer
surface, while preserving the good bulk properties. There are
Correspondence to: Changyou Gao, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China (e-mail: [email protected])
Contract grant sponsor: The Major State Basic Research Program of China; contract
grant number: G1999054305
© 2002 Wiley Periodicals, Inc.
838
many works devoted to the surface modification of PLA (or
PLLA) with an eye toward improving cytocompatibility.
Most of these works related the immobilization of natural
materials like gelatin,3 collagen,4 alginate,5 or biotin6 on the
hydrophobic PLA (or PLLA) surface.
Natural macromolecules such as gelatin, collagen, and
chitosan have been widely used as biomedical materials.7–9
These natural macromolecules have been successfully used
for a range of applications, including burn dressings, cardiovascular surgery, and 3D scaffolds for tissue engineering of
skin, bone, cartilage, etc.10 –15 Gelatin had been immobilized
on the PLLA surface by reacting the alkaline solution of
gelatin with PLLA directly. The modified PLLA showed
better cell attachment properties for 3T3 fibroblasts in vitro.3
Haw Suh had grafted Type I collagen on the ozone oxidized
PLLA membrane. The attachment, growth, and collagenous
protein synthesis of rat osteoblasts on the modified membranes were improved significantly in vitro.4 The success of
these natural materials may be attributed to the fact that they
are all naturally synthesized materials and they have good
hydrophilicity, good biodegradability, low immunogenicity,
and good cell attachment properties.
To improve the compatibility of PLLA for chondrocytes in
cartilage tissue engineering, a grafting and coating method
was developed to coat gelatin, collagen, or chitosan onto
PLLA surfaces. The natural macromolecules were chemically
grafted on the PLLA membrane surfaces as previously described.16 –-19 At the same time the natural macromolecule
solutions were physically coated on the grafted membranes to
IMMOBILIZATION OF NATURAL MACROMOLECULES
839
Figure 1. Schematic representation of the grafting and coating of the natural polymers on the PLLA
membrane surfaces. (a) Control PLLA membrane. (b) PLLA membrane with hydroperoxide groups on
the surface. (c) PLLA membrane with the grafted PMAA on the surface. (d) PLLA membrane with
activated carboxyl groups on the surface. (e) PLLA membrane with covalently grafted natural macromolecules on the surface. (f) PLLA membrane with grafted and coated gelatin on the surface. (g)
PLLA membrane with grafted and coated collagen or chitosan on the surface.
increase the surface density of the natural macromolecules.
The procedure is schematically shown in Figure 1. Experiment results showed that the coated layers adhered rather
tightly on the PLLA membranes and the adhesion, proliferation, and spreading of chondrocyte were improved obviously.
EXPERIMENT
Materials
The PLLA (Mn ⫽ 200,000, Mw ⫽ 400,000) was synthesized
using the method described in.20 The PLLA 1,4-dioxane
solution with a concentration of 6 wt.% was cast onto a
stainless steel plate and dried under vacuum to yield PLLA
membrane with a thickness of ⬇ 0.5 mm. Collagen I was
extracted from bovine tendons with the use of a previously
described method.21 Methacrylic acid (MAA, Shanghai
Chemical Industries Co. Ltd.) was purified by distillation
under vacuum. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, Aldrich), Chitosan (Aldrich), and gelatin
(Shanghai Chemical Industries Co. Ltd.) were used as received without further purification.
Grafting Copolymerization of Methacrylic Acid on the
PLLA Membrane Surface
Carboxyl groups were introduced onto the PLLA membrane
surface with the use of grafting copolymerization, as described previously16 –18 Briefly, the PLLA membrane [Figure
1(a)] was placed in hydrogen peroxide solution (30%) under
UV light at 50 °C for 40 min to introduce the hydroperoxide
groups onto the PLLA membrane surface. Then the photooxidized membrane [Figure 1(b)] was rinsed with deionized
water and immersed into methacrylic acid solution (5 vol%)
in a Pyrex glass tube purged with nitrogen. Grafting polymerization was carried out under UV irradiation at 50 °C for
60 min. The hydroperoxide groups were decomposed under
UV light to form macromolecular radicals P-O䡠 (P represents
PLLA), which can initiate the grafting copolymerization of
MAA on the PLLA membrane surface to yield PLLA-gPMAA [Figure 1(c)]. The grafted membrane was rinsed with
deionized water at 70° C for 24 h to remove the adsorbed
homopolymer.
Immobilization of the Natural Macromolecules on the
PLLA-g-PMAA Membrane
The —COOH residues on the membrane surface were activated at 0 °C for 4 h in phosphate-buffered saline solution
840
MA ET AL.
branes prepared above were rinsed with deionized water for
4 h at 37 °C to remove the EDAC, the acetic acid or the
unreacted glutaraldehyde, then dried under vacuum and
weighed (W). The surface density of the natural macromolecules on the PLLA membrane was defined as (W ⫺ W0)/S,
where W0 is the initial weight of the membrane and S is the
surface area of the membrane.
Membranes with only chemically grafted natural macromolecules on the surface were also prepared. The gelatingrafted membrane was taken out from the gelatin solution and
rinsed with deionized water at 37 °C for 2 days to get rid of
the adsorbed gelatin. The membrane was gently brushed with
a cotton tampon to aid the removal of the final trace of the
nongrafted (adsorbed) gelatin. For the collagen and chitosan
grafted membranes, the same method was used, except the
rinsing solution was 0.3% acetic acid.
Cell Culture
Chondrocytes were isolated from cartilage tissue of rabbit
ears (Japanese big ear white). Briefly, cartilage tissue obtained from the rabbit ears was cut into small pieces. Chondrocytes were isolated by incubating the cartilage pieces in
F-12 HAM’s (Hyclone) culture medium containing 0.2%
collagenase II (Sigma) at 37 °C for 6 h under stirring. The
chondrocytes were centrifuged, resuspended in F-12 HAMs
supplemented with 20% fetal calf serum (FBS), 300 mg/l
glutamine, 50 mg/l vitamin C, 100 U/ml penicillin and 100
U/ml streptomycin. The cell suspension was then seeded in
11-cm tissue culture dishes (Falcon, seeding density is 2⫻104
cells/cm2) and incubated in a humidified atmosphere of 95%
air, 5% CO2 at 37 °C. After a confluent cell layer was formed
(about 3– 4 days), the cells were detached with the use of
0.25% trypsin in PBS. Subsequently, the cells were resuspended in the supplemented culture medium as described
above, and used for the experiments. The cells used in this
work were taken from the same rabbit.
Cell Adhesion and Proliferation
Figure 2. XPS survey scan spectra of the PLLA membranes immobilized with (a) gelatin, (b) collagen, and (c) chitosan with the grafting
and coating method.
(PBS, pH ⫽ 4.5) containing EDAC (10 mg/ml). Then the
activated membrane was reacted with gelatin (4 mg/ml in
PBS, pH ⫽ 4.5), collagen (4 mg/ml in 0.3% acetic acid) or
chitosan (4 mg/ml in 0.3% acetic acid) solution for 24 h at 0
°C. In this stage EDAC acted as a condensing agent, promoting the condensation between —COOH and —NH2 to form
amide bond [Figures 1(c)–1(e)]. The membrane was then
taken out from the solution without washing, held vertical to
allow drainage of the liquid, and dried under vacuum. For the
membranes coated with gelatin, glutaraldehyde (2.5 vol%)
was used to cross link the gelatin layer [Figure 1(f)]. For the
membranes coated with collagen or chitosan, no glutaraldehyde treatment was used [Figure 1(g)]. The modified mem-
The control and modified PLLA membranes were placed on
the bottom of the tissue culture plates (Costar, 24 wells). Cell
adhesion rate was determined at 24 h, with a seeding density
of 6 ⫻ 104 cells/cm2. Before harvesting the adherent cells by
trypsinization, twice-gentle washings with PBS were performed. The cells were counted using a haemocytometer. The
adhesion rate was defined as the percentage of the number of
the cells adhered on the sample to the number of the cells
adhered on TCPS.
Cell proliferation rate was determined after culturing
for 96 h with a seeding density of 3 ⫻ 104 cells/cm2. The
cells were washed gently with PBS before counting. The
proliferation rate was defined as the ratio of the cell
number counted on the sample at 96 h to the seeding
number on that sample.
All data are presented as the mean values of four different
culture wells seeded with the cells of the same rabbit.
IMMOBILIZATION OF NATURAL MACROMOLECULES
841
Figure 3. C(1s) core-level scan spectra of pure gelatin, collagen, chitosan, the control and the
modified PLLA membranes prepared by the grafting and coating method. (a) Gelatin, (b) collagen, (c)
chitosan, (d) PLLA, (e) PLLA– gelatin, (f) PLLA– collagen; (g) PLLA– chitosan; (I) C—C; (II) C—O; (III)
C(AO)ONH; (IV) C(AO)OO.
Cell Activity by MTT Assay
Cell Morphological Assessment
Cell activity assay was performed after 96 h following the
seeding (seeding density 3 ⫻ 104). The culture medium was
removed and the cells washed gently with PBS. New culture
medium containing 200 ␮l MTT (3-(4,5-dimethyl)thiazol-2yl-2,5-dimethyl tetrazolium bromide, 5 mg/ml) was added to
each culture well and incubated for 4 h. The MTT was
reduced to formazan pigment by living cells. Finally, the
culture medium was removed and the cells washed gently
with PBS. Dimethyl sulphoxide (DMSO) was added. The
absorbance at 490 nm was measured. The cell activity was
defined as the ratio of the absorbance from the sample to
TCPS. TCPS seeded with the cells was set as positive control
and TCPS without the cells negative control.
After being fixed with 2.5% glutaraldehyde for 30 min, the
cells were stained with Giemsa (The Third Chemical Co. of
Shanghai, China). Microscopic observation of the cell morphology (⫻ 200)was carried out with the use of an inverted
microscope.
Surface Characterization of PLLA Membranes
The ATR-IR spectra were recorded by a Nicolet MagnaIR560 machine. XPS spectra were obtained on a ESCA LAB
Mark II spectrometer employing AlKagr; excitation radiation.
The take-off angle of the XPS was 30° degree. The charging
shift was referred to the C (1S) line emitted from the saturated
842
MA ET AL.
Figure 4. ATR-IR spectra of the control and modified PLLA membranes prepared by the grafting and coating method. (a) Control; (b)
PLLA– gelatin; (c) PLLA– collagen; (d) PLLA-chitosan. I—amide I, II—
amide II.
hydrocarbon at 285.0 eV. To calculate the atomic ratio of N
to C on the outermost layer of the modified PLLA membranes, the collecting factor of 1.77:1 was used.
Static water contact angle was obtained on a KRUSS
DSA10-MK machine. The sessile contact angle (SCA) was
determined by placing a drop of water (0.8 ␮l) on the surface
and recording the angle between the horizontal plane and the
tangent to the drop at the point of contact with the substrate.
Captive bubble contact angle (CBCA) was measured by
observing the air bubble in water at room temperature within
30 s after it came into contact with the material surface. For
both methods, each value was averaged from 15 times measurements.
The surface morphology of the PLLA membranes was
measured with the use of scanning electronic microscopy
(SEM) after overcoating with gold.
RESULTS AND DISCUSSION
Immobilization of the Natural Macromolecules
on PLLA Membrane
With the use of the grafting and coating method, layers of
gelatin, collagen, or chitosan can be formed on the PLLA
membrane surface with a surface density of 0.24 ⫾ 0.08
mg/cm2, 0.20 ⫾ 0.04mg/cm2 and 0.17 ⫾ 0.01 mg/cm2 (n ⫽
4) respectively. XPS spectra of the modified PLLA membranes were shown in Figure 2. Because there are no nitrogen
atoms in PLLA molecules, whereas there are abundant nitrogen atoms in molecules of gelatin, collagen, and chitosan, the
appearance of the N(1s) peaks in the spectra of the modified
membranes directly confirmed the existence of the natural
macromolecules. Figure 3 gave the C(1s) core level scan
spectra of the control PLLA, the natural macromolecule
coated PLLA and the pure natural macromolecules. The
spectra were fitted according to the known binding energies
of different carbon containing groups.5,22,23 The difference
between the spectra of the modified PLLA and the control
PLLA was consistent with the spectra of the pure natural
macromolecules. In the spectra of pure gelatin and collagen,
the peak IV (⬃289.0) were very low, whereas in PLLA
membranes coated with gelatin or collagen the peak IV were
much higher due to the large amount of carbon atoms
C(AO)OO in PLLA molecules. For pure chitosan and the
PLLA membrane coated with chitosan, the peak II (⬃287.0
eV) was very high because in the polysaccharide molecules
most of the carbon atoms connect to oxygen with a single
bond, of which the peak is at ⬃287.0 eV.5
ATR-IR spectra of the control and modified PLLA membranes were shown in Figure 4. The broad absorption at
3000 –3700 cm⫺1 was assigned to the stretching vibration of
O-H or N-H from —NH2, —CONH2, —OH, or —COOH
groups in gelatin, collagen, or chitosan. Another broad absorption at 1520 –1680 cm⫺1 was examined for the identification of amide groups in the natural macromolecules.24
SEM was used to observe the morphological differences
between the control and modified PLLA membranes (Figure
5). The surfaces of the gelatin-coated and chitosan-coated
membranes were smoother than the control membrane. There
were many collagen fibrils existed on the collagen-coated
membrane.
Stability of the Natural Macromolecule Layers
The word stable is used to mean that the coated biocompatible layers adhered rather tightly on the PLLA membranes
and were not easy to elute from the membrane surface. By
directly coating the natural macromolecule solutions on the
PLLA membranes, the layers formed on the membrane surfaces are not stable. Chitosan solution cannot spread on the
PLLA membrane surface due to the hydrophobicity of PLLA.
Even the gelatin solution and collagen solution can spread on
the untreated PLLA membrane surface to form a homogeneous solution layer, after the membrane dried, phase separation occurred. A very heterogeneous surface was formed on
the gelatin-coated membrane [Figure 6(a)]. The collagen
layer was very easy to detach from the substrate due to the
weak interaction between the collagen and the hydrophobic
PLLA [Figure 6(b)].
However, the biocompatible layers coated on the PLLA
surface with the use of the grafting and coating method were
rather stable. The grafted natural macromolecules had covalent chemical bonds with the PLLA membrane and strong
interaction with the coated natural macromolecules due to the
hydrogen bonds between each other, leading to the stabilization of the coated layer. After incubating in PBS (pH ⫽ 7.4)
at 37 °C for 48 h, no weight loss was detected on the
collagen- or chitosan-coated membranes. On the gelatincoated membrane, an obvious weight loss of 2.0 ⫾ 0.6 mg/ml
843
IMMOBILIZATION OF NATURAL MACROMOLECULES
Figure 5. Surface morphology of the control and the modified membranes prepared by the grafting
and coating method. (a) Control; (b) PLLA– gelatin; (c) PLLA– collagen; (d) PLLA– chitosan.
was found if the gelatin layer was not cross linked with
glutaraldehyde. However, the weight loss was decreased to
0.5 ⫾ 0.16 mg/ml after cross linking.
The stability of the natural macromolecule layers was
further confirmed by XPS. Table I listed the atomic ratio of N
to C (N/C) on the outermost layer of the natural macromolecule-coated membranes before and after incubating in PBS
(pH ⫽ 7.4) at 37 °C for 48 h. The N/C of the coated PLLA
films can be used to estimate the surface density of the natural
macromolecules.19 The N/C of pure gelatin, collagen, and
chitosan determined by XPS were 19.5%, 20.5%, and 12.4%,
respectively. From Table I it can be seen that the grafted and
coated membranes had almost the same N/C as the corresponding pure natural macromolecules. That means the mem-
brane surfaces were almost completely covered by a layer of
the natural macromolecules. After incubating in PBS (pH ⫽
7.4) at 37 °C for 48 h, the N/C on the cross-linked gelatincoated membrane decreased only slightly, while an obvious
decrease occurred on the uncross-linked gelatin-coated membrane. On the collagen- or chitosan coated membranes the
N/C did not change after incubating in PBS (pH ⫽7.4) at 37
°C for 48 h, suggesting that the biocompatible layers were
rather stable. This is because that collagen and chitosan are
insoluble in PBS solution with a pH value of 7.4.
Wettability
Static water contact angle was used to characterize the hydrophilicity of the control and modified PLLA membranes
844
MA ET AL.
Figure 6. Surface morphology of the membranes prepared by directly coating the (a) gelatin or (b)
collagen on the untreated PLLA membranes.
(Table II). The unmodified PLLA membrane showed a relatively higher hydrophobicity. After modification, the water
contact angle was decreased, indicating an increase of the
hydrophilicity of the natural macromolecule-coated PLLA
membranes. A larger degree of water contact angle reduction
was found for CBCA compared with SCA. This is because in
air the hydrophobic parts of the natural macromolecules tend
to accumulate near the membrane–air surface to reduce the
surface energy, whereas in water the coated natural macromolecules rearrange their formation to let the hydrophilic
groups face toward the water to reduce the interfacial energy
between the water and the natural macromolecule-coated
PLLA membranes.25
Biocompatibility
Table I showed that the N/C of the membranes with only
covalently grafted natural macromolecules on the surface
was rather low, indicating that the surface density of the
TABLE I. Atomic Ratio of N/C on the Outermost Layer of the
Control and Modified PLLA Membranes
With Only
With Grafted and After incubating
Grafted
in PBS
Coated
Natural
(pH ⫽ 7.4)
polymers on Natural Polymers
on the Surface at 37°C for 48 h
the Surface
(%)
(%)
(%)
PLLA
PLLA–gelatin
PLLA–gelatina
PLLA–collagen
PLLA–chitosan
a
0
5
5
2.2
1.7
Treated with glutaraldehyde
0
22.8
23.1
21.4
11.0
0
9.2
18.2
20.7
11.8
grafted natural macromolecules was very small.19 These
experiments showed that PLLA membranes with such
small amount of the natural macromolecules did not have
better cytocompatibility for chondrocyte than the control
(data are not shown). On the grafted and coated membranes, the N/C was greatly increased, which indicates
high surface densities of the natural macromolecules. The
cell adhesion, proliferation, and activity on the unmodified
PLLA and the grafted and coated PLLA membranes were
shown in Figure 7. All three modified membranes had
higher cell adhesion rate and cell activity than the control.
The gelatin- or collagen-coated membranes had higher cell
proliferation rate than the control. The collagen-coated
membrane had the best cell attachment, the best cell activity, and a rather high cell proliferation rate. Morphology
of chondrocytes on the control and modified membranes
was shown in Figure 8. At 24 h after seeding the cells were
round and spherical on the unmodified PLLA membrane.
On the modified membranes the cells were flat, polygonal
and more spread out, which is the normal shape of chondrocyte.26 Cell shape affects cell growth, gene expression,
extracellular matrix metabolism, and differentiation.27 The
spreading and polygonal morphology of chondrocytes on
TABLE II. Water Contact Angle of the Control and
Modified PLLA Membranes Prepared by the
Grafting and Coating Method
PLLA
PLLA–gelatina
PLLA–collagen
PLLA–chitosan
a
Sessile Contact
Angle (deg)
Captive Bubble
Contact Angle (deg)
85.0 ⫾ 2.0
69.4 ⫾ 4.3
68.6 ⫾ 3.2
60.9 ⫾ 3.1
71.0 ⫾ 1.6
31.2 ⫾ 2.2
34.3 ⫾ 2.0
45.1 ⫾ 5.0
Treated with glutaraldehyde
IMMOBILIZATION OF NATURAL MACROMOLECULES
845
the cell layer formed on the control PLLA membrane was
much easier to detach from the substrate than the others
(Figure 9). That means the modification also improved the
adhesion strength of the chondrocytes on the PLLA membranes.
CONCLUSIONS
Figure 7. Adhesion, proliferation, and cell activity of chondrocyte on
(a) TCPS, (b) control PLLA and PLLA membranes immobilized with (c)
gelatin, (d) collagen, and (e) chitosan.
the modified PLLA membranes indicates better cytocompatibility than the control. After four days of culture,
confluent cell layers were formed on all membranes, but
With the use of the grafting and coating method, gelatin,
collagen, or chitosan was immobilized on the PLLA membrane surface to form a stable biocompatible layer on the
PLLA surface. XPS, ATR-IR and surface-morphology
analysis verified the existence and the stability of the
biocompatible layers. The attachment, proliferation, activity, and morphology of the chondrocytes were improved on
the modified membranes compared with on the control
membrane. Take into account the stability and the cytocompatibility, the collagen-coated PLLA membranes had
the best results. The method developed in this study may
be used to modify three-dimensional PLLA scaffold to
provide an ideal scaffold for tissue engineering especially
for artificial cartilage.
Figure 8. Chondrocyte morphology, 24 h, seeding density 40,000/cm2. (a) TCPS; (b) control PLLA; (c)
PLLA– gelatin; (d) PLLA– collagen; (e) PLLA– chitosan. Magnification ⫻200.
846
MA ET AL.
Figure 9. Chondrocyte morphology, 96 h, seeding density 40,000/cm2. (a) TCPS; (b) control PLLA; (c)
PLLA– gelatin; (d) PLLA– collagen; (e) PLLA– chitosan. Magnification ⫻200.
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