Biodegradability and cell-mediated contraction of porous

collagen scaffolds: The effect of lysine as a novel

crosslinking bridge
Lie Ma, Changyou Gao, Zhengwei Mao, Jie Zhou, Jiacong Shen
Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
Received 15 August 2003; revised 6 July 2004; accepted 9 July 2004
Published online 15 September 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30170
Abstract: A novel crosslinking method was adopted to
modify the porous collagen scaffolds by using a water-
soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (EDAC) and N-hydroxysuccinimide (NHS) in
the presence of lysine, which functions as a crosslinking
bridge. In vitro biodegradation tests proved that in the pres-
ence of lysine the biostability of the EDAC crosslinked scaf-
folds was greatly enhanced. The biostability of the resultant
scaffolds was also elucidated as a function of the concentra-
tions of lysine and EDAC/NHS. Compared to the Col-DHT,
the ability of the Col-EDAC and the Col/Lys to resist cell-
mediated contraction (CMC) was greatly enhanced. Yet no
obvious difference between the Col-EDAC and the Col/Lys
was found with respect to CMC. SEM observations showed
that the microstructure of the crosslinked scaffolds could be
largely preserved after broblast seeding. As a result, MTT
assays proved that the broblasts in the Col/Lys scaffolds
proliferated faster compared to the DHT-treated one on the
assumption that the cell viability was preserved to a similar
level. Histological section results indicated that the Col/Lys
scaffolds had the ability to accelerate the cell inltration and
proliferation. All these results demonstrated that this novel
crosslinking method is an effective way to achieve a collagen
scaffold with improved biostability and a more stable struc-
ture, which can resist cell-mediated contraction. 2004
Wiley Periodicals, Inc. J Biomed Mater Res 71A: 334342,
2004
Key words: collagen; scaffold; lysine; crosslink; contraction;
tissue engineering
INTRODUCTION
Because of the excellent biocompatibility of colla-
gen, the use of porous collagen scaffolds as a regen-
eration template for tissues such as skin,
13
cartilage,
4,5
bones,
6
and nerve
7,8
has been widely researched.
However, the fast biodegradation rate and low me-
chanical strength of the untreated collagen cannot
meet the demands of in vitro or in vivo applications in
many cases, and have been the crucial factors that
limit the further use of this material.
9,10
Crosslinking of the porous collagen scaffolds is an
effective way to slow the biodegradation rate and
optimize the mechanical properties.
11,12
Currently,
two kinds of crosslinking methods are frequently em-
ployed to improve the properties of the porous colla-
gen scaffoldsphysical methods and chemical meth-
ods. The former includes the use of photooxidation,
dehydrothermal treatments (DHT), and ultraviolet ir-
radiation, which can avoid introducing potential cyto-
toxic chemical residuals and sustain the excellent bio-
compatibility of collagen materials.
13
However, most
of the physical treatments cannot yield enough
crosslinking to meet the demands of tissue engineer-
ing. Therefore, treatments by chemical methods are
still necessary in most cases. Several crosslinking
agents, such as glutaraldehyde (GA), 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide (EDAC), poly-
glycidyl ether, and polyepoxides have been used
widely.
11,1417
Among them, the EDAC/N-hydroxy-
succinimide (NHS) crosslinking system, which has
shown better biocompatibility than GA, is used fre-
quently.
12
This crosslinking takes place by reaction
between carboxyl groups of glutamic or aspartic acid
residues and amine groups to form amide bonds.
1821
However, the crosslinking degree cannot match the
applications of tissue engineering in many cases under
the EDAC/NHS treatment. Therefore, diamine agents
such as putrescine, hexane diamine, and so forth, have
Correspondence to: C. Gao; e-mail: cygao@mail.hz.zj.cn
Contract grant sponsor: Natural Science Foundation of
China; contract grant number: 50173024
Contract grant sponsor: Science and Technology Program
of Zhejiang Province; contract grant number: 2004C21022
Contract grant sponsor: Major State Basic Research Pro-
gram of China; contract grant number: G1999054305
2004 Wiley Periodicals, Inc.
been used to assist the crosslinking.
15,22
Up to now the
use of amino acids as crosslinking bridges to enhance
the biostability of porous collagen scaffolds has rarely
been reported. Amino acids are the basic components
of proteins and naturally have good biocompatibility.
A preliminary study has found that amino acids with
neutral, acidic, or basic properties have different ef-
fects on the crosslinking efciency. Basic amino acids
are favorable for the improvement of biostability.
The contraction of cell-seeded matrices and in vivo
implants is a key factor that inuences the construc-
tion of tissue engineering organs. Because of the con-
traction of cell-seeded scaffolds, changes in the size
and microstructure of the implants take place fre-
quently. A change in size will cause difculty in tting
a specic implant site, or cause separation from the
surrounding tissue host.
23,24
The crucial effect of the
contraction is that the pore size and porosity reduction
of the scaffolds will impede the migration of cells from
the surrounding tissue into the scaffolds and affect the
free exchange of the nutriments and the metabolites.
Contraction of collagen-based matrices, such as gels
and scaffolds, caused by many types of connective
cells, has been reported in many studies.
25
In order to
resist the contraction, crosslinking treatments are ap-
plied to enhance the mechanical properties of the scaf-
folds.
In this study, a novel crosslinking strategy, that is,
EDAC/NHS in the presence of lysine, was em-
ployed to improve the biostability and the ability to
resist cell-mediated contraction of porous collagen
scaffolds. The factors controlling the biostability of
the crosslinked scaffolds (Col/Lys) and the effect on
the cell-mediated contraction were researched in
detail. Changes to the microstructure and the cyto-
compatibility of the scaffolds were evaluated as
well.
MATERIALS AND METHODS
Materials
Collagen type I was isolated from fresh bovine tendon by
a trypsin digestion and acetic acid dissolution method.
26
Lysine (Lys) and hydroxyproline were purchased from
Shanghai Amino Acid Company (China). Glutaraldehyde
(GA), 25% water solution, was a product of Shanghai Phar-
maceutical Company (China). Collagenase (278 U/mg),
trypsin (250 U/mg), 1-ethyl-3-(3-dimethylaminopropyl)-car-
bodiimide (EDAC), N-hydroxysuccinimide (NHS), and
2-morpholinoethane sulphonic acid (MES) were purchased
from Sigma. All other reagents and solvents were of analyt-
ical grade and were used as received.
Preparation of porous collagen scaffold
Collagen was dissolved in 0.5M acidic acid solution to
obtain a 0.5% (w/v) solution. After being deaerated under
reduced pressure to remove entrapped air bubbles, the col-
lagen solution was injected into a homemade mold (diame-
ter: 16 mm, depth: 2 mm), frozen in 70% ethanol bath at
20C for 1 h, and then lyophilized for 24 h to obtain a
porous collagen scaffold.
Crosslinking treatments
All the scaffolds were dehydrated at 105C under vacuum
(less 0.2 mbar, DHT) for 24 h prior to any additional chem-
ical crosslinking. Collagen scaffolds weighted 2.5 mg were
incubated in 1 mL 50 mM MES (pH 5.5) solution containing
(Col/Lys) or not containing (col-EDAC) 2.5 M lysine for
1 h at room temperature. Then 1 mL MES solution contain-
ing 40 mM EDAC and 20 mM NHS was added. After incu-
bation for 24 h at room temperature, the scaffolds were
washed with double-distilled water (10 min 6 times) and
lyophilized.
The porous collagen scaffolds were also crosslinked with
20 mM GA in the presence or absence of lysine in order to
compare the crosslinking efciency of these two kinds of
crosslinking agents.
Characterization of collagen scaffolds
The microstructure of the scaffolds before or after
crosslinking with EDAC/NHS in the presence or absence of
lysine was observed under scanning electron microscopy
(SEM, Cambridge stereoscan 260). The mean pore size of the
scaffolds was determined by analysis of the SEM images
from the cross-section positions. The porosity of each kind
scaffold was measured by immersing the scaffolds (initial
weight, W
0
) into ethanol at room temperature for 24 h (wet
weight, W) and calculated by the formulas below.
porosity (%)
(WW
0
)
1

1
W
2

1
W
0
100%,
where
1
and
2
represent density of collagen (1.21 g/mL)
and ethanol (0.79 g/mL), respectively.
The swelling test was performed by incubating the scaf-
folds in distilled water at room temperature and determin-
ing the wet weight of the scaffolds 24 h later. The swelling
ratio of the scaffolds was dened as the ratio of weight
increase to the initial weight. Each value was averaged from
three parallel measurements.
In vitro collagenase degradation
The biological stability of the crosslinked collagen scaf-
folds was evaluated by in vitro collagenase biodegradation
test. Each kind of scaffold was incubated in phosphate-
LYSINE AS A NOVEL CROSSLINKING BRIDGE 335
buffered saline (PBS, pH 7.4) containing a given concentra-
tion of collagenase (type I, Sigma) at 37C. The degradation
was terminated at the given time interval by incubating the
assay mixture in an ice bath immediately. Following centrif-
ugation at 1000 rpm for 10 min, the clear supernatant was
hydrolyzed with 6M HCl at 120C for 12 h. The amount of
hydroxyproline released from the collagen molecules in the
scaffolds was measured by absorbance spectroscopy (CARY
100 BIO, America) at a wavelength of 560 nm.
27
The biodeg-
radation degree is dened as the percentage of the released
hydroxyproline from the scaffolds to the completely de-
graded one with the same composition and weight.
Fibroblast isolation and scaffold seeding
Fibroblasts used in this study were isolated from human
dermis by collagenase digestion.
28
The cell suspensions
were cultured at 37C, 5% CO
2
, and 95% humidity in DMEM
supplemented with penicillin (100 U/mL), streptomycin
(100 U/mL), and 10% fetal bovine serum (FBS) (complete
medium), which was changed every 3 days.
Cells were passaged at conuence and the fourtheighth
passage broblasts were used for the in vitro seeding. The
scaffolds were sterilized in 75% ethanol for 12 h, followed
with solvent exchange by PBS six times. Each scaffold was
then placed on a 24-well polystyrene plate (Falcon) and
seeded with 200 L human dermal broblast suspension at
a density of 5 10
6
cells/mL. After incubation for 4 h, the
cell-seeded scaffold was moved to another well in the 24-
well plate with 1 mL complete medium. Then the scaffolds
were cultured in a 5% CO
2
incubator at 37C with medium
change every 3 days. Cultures were terminated 3, 7, 14, and
21 days after seeding. The unseeded scaffolds were incu-
bated in the same culture conditions and employed as con-
trols.
Measurement of the scaffold contraction
The diameters of the seeded and unseeded scaffolds were
determined with a ruler with 0.5-mm intervals (n 3). The
contraction of the scaffolds was calculated by dividing the
diameter change of each scaffold at the termination of the
culture time by its original diameter. Herein, cell-mediated
contraction (CMC) was determined by subtracting the con-
traction of the unseeded scaffolds from the contraction of the
seeded scaffolds.
23,29
CMC

original diameter diameter
original diameter

Seeded

original diameter diameter
original diameter

Unseeded
.
Morphology of the fibroblast-seeded scaffold
At 7 and 21 days after seeding, the culture was discontin-
ued and the scaffolds were washed with PBS and xed with
2.5% glutaraldehyde at room temperature for 24 h. After
being washed with PBS to remove the remaining glutaral-
dehyde, the scaffolds were dehydrated with a graded series
of ethanol. Then the scaffolds were further dehydrated with
acetone and treated with isoamyl acetate. After being dried
by the critical point dry method, the scaffolds were coated
with an ultrathin gold layer and observed under scanning
electron microscopy (SEM, Cambridge stereoscan 260).
Behavior of cell proliferation
The cell proliferation as a function of culture time was
evaluated by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetra-
zoliumbromide (MTT) assay according to the methods of
Mosmann with minor modication.
30
At each culture inter-
val, the media in the plates were removed and 200 L MTT
(5 mg/mL, Sigma)/complete medium (volume ratio 1:4)
solution was injected into the scaffolds. After being incu-
bated for 4 h, the scaffolds were transferred into a ask,
where dimethylsulfoxide (DMSO) was added up to 500 L.
Then the solution was mixed thoroughly with a vortex
mixer. Finally, the absorbance of violate substance was mea-
sured at 570 nm by using a microplate reader (Biorad, model
550).
Histological observation
At the termination of the cultures, the cell-seeded Col/Lys
scaffolds were xed with 10% neutral buffered formalin at
room temperature for 24 h and embedded in parafn. The
specimens were cross sectioned at a thickness of 5 m and
stained with hematoxylin and eosin (H&E). The morphology
and distribution of cells in the scaffolds were observed with
a light microscope (Axiovert 200, Zeiss)
RESULTS
Microstructure and swelling property
Figure 1 shows the cross-section morphology of the
scaffolds before and after crosslinking treatment. The
cross-section images reveal that the porous structure
of the collagen scaffolds was largely preserved after
crosslinking either in the presence or absence of lysine.
However, more sheet-like structures could be ob-
served after crosslinking. Table I lists the analyzing
data on the scaffold pore size, porosity, and PBS swell-
ing ratio. As seen in the table, the typical pore size is
smaller than 100 m in the Col-DHT scaffolds. In
either the presence or absence of lysine, the mean pore
size became slightly enlarged under the EDAC/NHS
treatment. No signicant changes in the porosity were
observed before or after the crosslinking treatment.
All the scaffolds have porosities larger than 99%.
336 GAO ET AL.
However, the crosslinking leads to an obvious de-
crease in swelling ratios. The value was lowered from
105.9 6.2 for the Col-DHT, to 79.3 4.6 for the
Col-EDAC, and further to 68.8 1.9 after crosslinking
in the presence of lysine (Col/Lys).
In vitro biodegradation
Figure 2 compares the biodegradation degree of the
scaffolds by the collagenase digest test. The gure
shows that the Col-DHT has been fully degraded after
incubation in the collagenase solution for 12 h. After
Figure 1. SEM images to show the cross sections of the collagen scaffolds treated with EDAC/NHS in the presence/absence
of lysine (original magnication 200). (a) Col-DHT, (b) Col-EDAC, (c) Col/Lys.
Figure 2. The effect of lysine on the biodegradation degree
of the collagen scaffolds crosslinked with EDAC/NHS or
GA. All scaffolds were incubated in 100 g/mL (30 units)
collagenase for 12 h. Values are mean standard deviation
(n 3).
TABLE I
Comparison of the Microstructure Parameters and
Swelling Ratio between the Col-DHT and the
Cross-Linked Scaffolds
Pore Size
(m) Porosity
Swelling
Ratios
Col-DHT 99 19 99.46 0.02% 105.9 6.2
Col-EDAC 106 17 99.35 0.03% 79.3 4.6
Col/Lys-EDAC 107 16 99.44 0.03% 68.8 1.9
LYSINE AS A NOVEL CROSSLINKING BRIDGE 337
being treated with EDAC/NHS, the crosslinked colla-
gen scaffold (Col-EDAC) was more resistant to colla-
genase digestion, and the biodegradation degree was
decreased from 100% to 23.3 4.3%. With the addi-
tion of lysine (Col/Lys), a lower biodegradation de-
gree, 9.1 2.4%, was achieved. Compared to the value
of Col-EDAC, the decrease in the biodegradation de-
gree was highly signicant (P 0.007). The biodegra-
dation degree of the Col-GA and the Col/Lys-GA was
7.7 4.6% and 6.7 2.8%, respectively, after the same
digestion procedure. No signicant difference was de-
tected between these two scaffolds (P 0.76). The
Col-GA showed a lower biodegradation degree com-
pared to Col-EDAC (P 0.01). The addition of lysine
to the scaffolds crosslinked with EDAC/NHS resulted
in biostability similar to that of the GA crosslinked
material.
The effect of the lysine concentration on biostability
is illustrated in Figure 3. The biodegradation degree
decreased as a function of the concentration of lysine
when the concentration was lower than 20 M. From
0 to 10 M, the biodegradation degree decreased
sharply with the increase of lysine concentration. Then
the biodegradation degree had a slight decrease when
the lysine concentration was increased to 20 M. With
further increase of the lysine concentration, the bio-
degradation degree was kept nearly constant up to 100
M.
Figure 4 shows the biodegradation behavior of the
scaffolds crosslinked with different concentrations of
EDAC/NHS in the presence of 2.5 M lysine. When
the EDAC and NHS concentrations were 5 and 2.5
M, respectively (abbreviated as 5/2.5), the scaffold
had been degraded 26.4% over the 12-h digestion pe-
riod. After 96 h of digestion, the biodegradation
degree was increased to 60%. The degradation rate
became slower with increasing EDAC/NHS concen-
tration. Only 9.9% of scaffolds were degraded after
digestion for 96 h when the concentrations of EDAC
and NHS were 50/25. It is worth noting that concen-
tration increase higher than 30/15 has no signicant
effect on the decrease of the biodegradation degree.
Scaffold contraction
The cell-mediated contraction (CMC) of the scaf-
folds as a function of culture time is shown in Figure
5. For the Col-DHT, the CMC value had increased to
0.34 after culture for only 3 days. Twenty-one days
Figure 3. The biodegradation degree of the collagen scaf-
folds as a function of the concentrations of lysine under the
treatment of EDAC/NHS (20/10). All scaffolds were incu-
bated in 500 g/mL (150 units) collagenase for 12 h.
Figure 4. The biodegradation degree of the collagen scaf-
folds as a function of the concentration of EDAC and NHS in
the presence of 2.5 M lysine. All scaffolds were incubated
in 100 g/mL (30 units) collagenase solutions.
Figure 5. Cell-mediated contraction (CMC) of the Col-
DHT and Col/Lys scaffolds as a function of the culture time.
Contraction was normalized to the shrinkage of the un-
seeded scaffolds. Values are mean standard deviation (n
3).
338 GAO ET AL.
later, the CMC value was 0.7, meaning that the scaf-
fold had been largely contracted. In contrast to the
Col-DHT, no obvious contraction was observed for the
Col-EDAC and Col/Lys scaffolds during the 21-day
culture period. The addition of lysine has no signi-
cant enhancement to resist the cell-mediated contrac-
tion.
The cross-section images of the scaffolds after 21
days of culture are presented in Figure 6. In compar-
ison to Figure 1, the pores in the Col-DHT are hardly
distinguished at the same magnication [Figure 6(a)].
It indicates that the pore size was reduced to a large
extent because of the cell-mediated contraction. How-
ever, the three-dimensional structure of the Col/Lys
was still preserved with a pore size similar to that of
the unseeded one because of the crosslinking treat-
ment [Figure 6(b)].
Cytocompatibility
Figure 7 shows the cell-proliferation behavior by
MTT assay. For the Col-DHT, the cell viability in the
scaffold increased in the rst week. Then, with pro-
longed culture time, the cell viability decreased. How-
ever, the cell viability of the Col/Lys increased over
the whole culture period, and the Col/Lys always
behaved with higher viability than the Col-DHT (P
0.005 at all intervals).
The SEM images of the cell-seeded Col/Lys scaf-
folds are displayed in Figure 8. After being cultured
for 7 days, several broblasts could be detected in the
scaffold with a typical shuttle-like morphology [Fig-
ure 8(a)]. When the culture time was prolonged, more
cells were observed in the scaffold. The cells spread
well along the collagen bers and had been conuent
to some extent [Figure 8(b)]. Histological results show
that the cells only existed at the edge of the scaffold
during the initial culture stage. After being cultured
for 21 days, many broblasts could be observed both
on the surface and in the inside of the scaffold and
distributed evenly (Figure 9). This proves that the cells
had inltrated into the Col/Lys and proliferated there.
DISCUSSION
The effects of the lysine used as a novel crosslinking
bridge on enhancing the ability of collagen scaffolds to
resist the collagenase biodegradation and the cell-me-
diated contraction are demonstrated. It is well known
that the three-dimensional porous structure of a scaf-
fold is rather important for cell inltration and prolif-
eration.
31
Therefore, the preservation of the porous
structure is a crucial aspect to evaluate a crosslinking
method. In general, the crosslinking treatment always
Figure 6. SEM images showing the cross sections of (a) the Col-DHT and (b) the Col/Lys scaffolds after culturing for 21
days.
Figure 7. The comparison of the cell viability between the
Col-DHT and the Col/Lys scaffolds at different culture ter-
minations. Values are mean standard deviation (n 3).
LYSINE AS A NOVEL CROSSLINKING BRIDGE 339
results in some extent of collapse because of the rehy-
dration process. After crosslinking by EDAC/NHS
under the assistance of lysine, the porous structures
were largely preserved, with a slight reduction in
porosity compared to the Col-DHT one. Meanwhile,
the crosslinking treatments have some effect on en-
larging the pore size. These results can be explained
by the recombination of the collagen bers during the
rehydration and refreezedrying procedures of the
crosslinking treatment.
28,32
Swelling ratio is another key factor of a scaffold,
representing the ability to hold the nutriments to ac-
celerate cell inltration and proliferation as well as to
transfer the metabolites through the scaffold.
33
The
crosslinking treatment led to about 30% shrinkage in
swelling ratio. This decrease should be mainly attrib-
uted to the partial diminishing of the hydrophilic
groups and the collapse of the scaffold after crosslink-
ing. The initially existing hydrophilic groups, such as
amino groups and carboxyl groups, were partially
transformed into hydrophobic amide groups under
the EDAC/NHS treatment. Nevertheless, the swelling
ratio of the crosslinked scaffolds is still big enough to
meet the demand of the matrices used in tissue engi-
neering.
To the best of the authors knowledge, a higher
crosslinking efciency can be achieved under GA
treatment than under EDAC/NHS treatment. How-
ever, EDAC/NHS has been used more frequently in
the treatment of porous collagen scaffolds, because of
better biocompatibility. In this study, the collagen
scaffolds crosslinked by EDAC/NHS in the presence
of lysine had biostability similar to that of the GA
crosslinked one. The added lysine could function as a
crosslinking bridge between collagen molecules. Pre-
vious study has proved that the biodegradation de-
gree depends on the NH
2
/COOH ratio in the EDAC/
NHS crosslinking system. In the presence of amino
acids, there are two complete reactions. One is the
functional groups blocking reaction by amino acids;
the other is the bridging reaction between two colla-
gen molecules. The predominant reaction can be tai-
lored by controlling the total NH
2
/COOH ratio in the
crosslinking system. Below an optimal ratio, the bio-
degradation degree decreases with augmented NH
2
/
COOH ratios; then a reversed function can be ob-
tained. Therefore, the further addition of lysine results
in the NH
2
/COOH ratio closing to a constant, for
which the biodegradation degree tends to be a con-
stant with increasing lysine concentration. It is easy to
understand that the biodegradation degree will de-
crease with increasing EDAC/NHS concentration at
the beginning. When EDAC/NHS is too excessive,
further addition will have little effect on the crosslink-
ing degree.
Along with enhanced biostability, the crosslinking
treatment yielded a more stable structure that can
Figure 8. SEMimages of the morphology of broblasts in the Col/Lys scaffolds after culturing for (a) 7 days and (b) 21 days.
Figure 9. Light micrograph of the H&E-stained sections of
the broblast-seeded Col/Lys scaffolds at 21 days (original
magnication, 200). Bar indicates 100 m.
340 GAO ET AL.
better resist cell-mediated contraction. The scaffolds
contraction may be due to the contraction of the myo-
broblasts, a major contractile phenotype of bro-
blasts that expresses a smooth muscle actin isoform,
-smooth muscle actin (SMA).
34
In general, the con-
tractile cells are inevitable in the seeding population,
because of the process of the cell isolation and the
two-dimensional culture conditions.
35
Hence, the ob-
vious contraction always occurred in the Col-DHT
collagen matrix implanted in vitro or in vivo for its low
mechanical properties. Although the addition of ly-
sine could bring a great enhancement in biostability,
there is no obvious difference in the ability to resist
contraction between the Col-EDAC and the Col/Lys.
This would mean that the contractile force caused by
SMA is not big enough to distinguish the difference of
CMC between these two kinds of scaffolds.
The effects of the contraction will reduce the pore
diameter of the scaffolds and compress the space for
cell proliferation and the diffusion of the nutrients.
23
It
is important to determine the successive proliferation
of the seeding broblasts. In order to evaluate cyto-
compatibility of the scaffolds, the MTT assay was ap-
plied to analyze the cell proliferation because the ab-
sorbance value in 570 nm can be correlated to the cell
number in the scaffolds on the assumption that the cell
viability was preserved to a similar level.
33
When the
culture time was prolonged, the viability of the Col/
Lys increased gradually. It can be observed that the
violating substance was only on the surface in the rst
week, then on the entire scaffold after 21 days of
culturing. The low CMC of the Col/Lys resulted in the
three-dimensional structure being largely preserved.
Therefore, the Col/Lys scaffolds have enough space
for the cell proliferation. In contrast, the Col-DHT
scaffolds contracted 50% in the rst week, so that the
cell viability did not increase further, but decreased
with increasing culture time. This can be explained by
the limited amount of available nutriments and the
limited amount of available space caused by the con-
traction. Meanwhile, both the SEM images and the
histological sections indicated that the broblasts
could spread along the wall of the pores and prolifer-
ate well in the scaffolds. It is attributed to the original
cytocompatibility of lysine, the addition of which does
not deteriorate the excellent cytocompatibility of col-
lagen. All these results prove that this novel crosslink-
ing strategy is an effective way to modify the porous
collagen scaffold.
CONCLUSIONS
Herein lysine is applied as a novel crosslinking
bridge to enhance the biostability of the porous colla-
gen scaffolds. Under the crosslinking treatment, the
microstructures of the scaffolds were largely pre-
served. Biostability was much improved after
crosslinking in the presence of lysine, resulting in a
similar crosslinking degree as the GA crosslinked one.
There exists an optimal concentration of lysine and
EDAC/NHS to increase the biostability. Compared to
the Col-DHT, the ability of the Col-EDAC and the
Col/Lys to resist the cell-mediated contraction was
greatly enhanced. Yet no obvious difference between
the Col-EDAC and the Col/Lys was found with re-
spect to CMC. A porous structure could remain after
21 days of culture, which could accelerate the cell
inltration. Cell MTT assay and histological results
proved that the cells could proliferate well and dis-
tribute evenly in the Col/Lys scaffolds. In conclusion,
crosslinking by EDAC/NHS in the presence of lysine
is an effective method to fabricate collagen scaffolds
with enhanced biostability and good cytocompatibil-
ity.
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